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USP7 targets XIAP for cancer progression: Establishment of a p53-independent therapeutic avenue for glioma

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Gouranga Saha at Regional Centre for Biotechnology
Gouranga Saha
Sibani Sarkar at Indian Institute of Chemical Biology
Sibani Sarkar
Partha S. Mohanta
Partha S. Mohanta
Partha S. Mohanta
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Krishna Kumar at Indian Institute of Chemical Biology
Krishna Kumar
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Abstract and Figures

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the ‘USP7-Mdm2-p53’ axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.
P53 independent apoptosis uponUSP7 inhibition: XIAP is a possible mediator A HCT116 (p53Wt and p53Null) lines were treated with P5091 (0 to 80 µM) for 24 and 48 h and cell viabilities were checked by MTT assay. The inset graph represents the mean IC-50 values of both cells at respected time points. B Indicated protein levels were determined by immunoblotting (IB), where HCT116 (p53Wt and p53Null) cell lines were treated with P5091(15 µM) for 24 h. GAPDH was kept as the loading control. C HCT116 (p53Wt and p53Null) cells were treated with different doses of P5091 for 24 h, the increase in caspase 3/7 activity was determined using Caspase-Glo® 3/7 Assay kit. D Number of colonies formed by HCT116 (p53Wt), HepG2 (p53Wt), and Huh7 (p53Mut) cells after treatment with P5091 (15 µM) for 24 h. E, F Silver stained SDS-PAGE gels containing elute from respective pull-down as indicated. Protein bands were used to identify USP7 and XIAP interacting proteins by Mass Spectrometry using HEK293 cell lysates. G Over representation analysis (ORA) of USP7 interactome revealed enriched pathways as par Reactome 2016 library. H Volcano plot represents significantly up-and down-regulated proteins at FDR 0.05 and FC of 1.5. Proteins associated with the Apoptosis and Reactive Oxygen Species (ROS) generation are indicated in the plot. I Heatmap of significantly regulated proteins protein following hierarchical clustering (by Perseus) based on Euclidean distance display the top 10 regulated proteins from each cluster. J, K GSEA analysis of all quantified proteins depicts significantly enriched phenotypes in Control and P5091 groups. L Analysis of NCI-60 human tumor cell lines for USP7 and XIAP mRNA (z-score expression) level correlation analysis from GSE32474, and correlation analysis of USP7 and XIAP normalized protein level from PXD013455. [Error bars in all the indicated sub-figures represent mean ± SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 and represented as asterisk (*), otherwise nonsignificant (ns)].
P53 independent apoptosis uponUSP7 inhibition: XIAP is a possible mediator A HCT116 (p53Wt and p53Null) lines were treated with P5091 (0 to 80 µM) for 24 and 48 h and cell viabilities were checked by MTT assay. The inset graph represents the mean IC-50 values of both cells at respected time points. B Indicated protein levels were determined by immunoblotting (IB), where HCT116 (p53Wt and p53Null) cell lines were treated with P5091(15 µM) for 24 h. GAPDH was kept as the loading control. C HCT116 (p53Wt and p53Null) cells were treated with different doses of P5091 for 24 h, the increase in caspase 3/7 activity was determined using Caspase-Glo® 3/7 Assay kit. D Number of colonies formed by HCT116 (p53Wt), HepG2 (p53Wt), and Huh7 (p53Mut) cells after treatment with P5091 (15 µM) for 24 h. E, F Silver stained SDS-PAGE gels containing elute from respective pull-down as indicated. Protein bands were used to identify USP7 and XIAP interacting proteins by Mass Spectrometry using HEK293 cell lysates. G Over representation analysis (ORA) of USP7 interactome revealed enriched pathways as par Reactome 2016 library. H Volcano plot represents significantly up-and down-regulated proteins at FDR 0.05 and FC of 1.5. Proteins associated with the Apoptosis and Reactive Oxygen Species (ROS) generation are indicated in the plot. I Heatmap of significantly regulated proteins protein following hierarchical clustering (by Perseus) based on Euclidean distance display the top 10 regulated proteins from each cluster. J, K GSEA analysis of all quantified proteins depicts significantly enriched phenotypes in Control and P5091 groups. L Analysis of NCI-60 human tumor cell lines for USP7 and XIAP mRNA (z-score expression) level correlation analysis from GSE32474, and correlation analysis of USP7 and XIAP normalized protein level from PXD013455. [Error bars in all the indicated sub-figures represent mean ± SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 and represented as asterisk (*), otherwise nonsignificant (ns)].
… 
XIAP: a putative substrate of USP7 A HEK293 cells were transfected with EV, pGZ-USP7, pGZ-USP7C223S, and three different shRNAs against USP7 along with scramble shRNA as depicted in the figure. IB was performed 48 h post-transfection for USP7 and XIAP expression. B qRT-PCR analysis showing XIAP mRNA fold change upon USP7 inhibition by P5091, USP7 knockdown and over-expression. XIAP and USP7 protein level from the same experiment represented by IB. C Multiple cancer cell lines as indicated treated with P5091 (15 µM) for 24 h and XIAP level was analyzed by IB. table represent the origin and p53 mutational status of cell lines used. D IB analysis of XIAP was performed in HCT116 (p53Wt) cells transfected with GFP- USP7, pGZ-USP7C223S, or siUSP7. EV and siControl were kept for respective controls. E HCT116 cells were transfected with either siControl or siUSP7, and USP7 was inhibited with P5091 (10 µM) for 24 h (as depicted in the Figure, upper and lower panels) before treatment with cycloheximide (CHX: 50µG/ml). XIAP protein levels at indicated time points were analyzed by IB. Graph showing the change in XIAP half-life. [GAPDH was used as an internal loading control in all IB experiments. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 is represented as asterisk (*), otherwise nonsignificant (ns)].
XIAP: a putative substrate of USP7 A HEK293 cells were transfected with EV, pGZ-USP7, pGZ-USP7C223S, and three different shRNAs against USP7 along with scramble shRNA as depicted in the figure. IB was performed 48 h post-transfection for USP7 and XIAP expression. B qRT-PCR analysis showing XIAP mRNA fold change upon USP7 inhibition by P5091, USP7 knockdown and over-expression. XIAP and USP7 protein level from the same experiment represented by IB. C Multiple cancer cell lines as indicated treated with P5091 (15 µM) for 24 h and XIAP level was analyzed by IB. table represent the origin and p53 mutational status of cell lines used. D IB analysis of XIAP was performed in HCT116 (p53Wt) cells transfected with GFP- USP7, pGZ-USP7C223S, or siUSP7. EV and siControl were kept for respective controls. E HCT116 cells were transfected with either siControl or siUSP7, and USP7 was inhibited with P5091 (10 µM) for 24 h (as depicted in the Figure, upper and lower panels) before treatment with cycloheximide (CHX: 50µG/ml). XIAP protein levels at indicated time points were analyzed by IB. Graph showing the change in XIAP half-life. [GAPDH was used as an internal loading control in all IB experiments. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 is represented as asterisk (*), otherwise nonsignificant (ns)].
… 
XIAP and USP7 colocalize and physically interact A Representative image of immunofluorescence staining of USP7 (green), XIAP (red) in HeLa, HCT116, and A549 cells. Images were captured at 60X optical magnification; Scale bar represents 20 µm. B Purified Myc-His-USP7 incubated separately with GST and GST-XIAP in the presence of GSH- beads for 4 h. SDS-PAGE separated samples were stained with Coomassie blue to visualize the specific bands for GST, GST-XIAP, and Myc-His USP7. C HEK293 cell lysate was incubated separately with purified GST and GST-XIAP in the presence of GSH-beads for 4 h, followed by IB using indicated antibodies. D HEK293 cell lysate was immunoprecipitated with USP7 antibody followed by IB using indicated antibodies. E Interaction in overexpressed conditions: HEK293 cells were transiently transfected with FLAG-USP7 and GFP-XIAP or GFP as the control, followed by immunoprecipitation with anti-Flag antibody, and IB was performed with antibodies against Flag and GFP. F HEK293 cells were treated with either DMSO or MG132 (25 µM) for 8 h. Equal amounts of lysates were used for IP using USP7 antibody followed by IB with indicated antibodies. Veriblot secondary antibody was used to prevent nonspecific detection of the heavy and light chains. G Venn diagram shows a comparison of top-six interacting domain pairs of USP7 and XIAP obtained using three different docking software. H Upper panels showing full-length USP7 and XIAP proteins generated by stitching the available USP7 and XIAP domains in PDB. Lower panels were showing USP7-XIAP complex, where interface region was highlighted in yellow. I Schematic representation of USP7 deletion mutants interacting with full-length XIAP. J HEK293 cells were co-transfected with indicated plasmids; cell lysates were pull-down with Ni-NTA beads for 1 hr at room temperature. Pulled-downed and input were analyzed by IB with the indicated antibodies. Ponceau S staining indicates equal loading. K Schematic representation of XIAP deletion mutants interacting with full-length USP7. L HEK293 cells were transiently transfected with Flag-USP7. The lysates were incubated separately with GST or GST-XIAP deletion mutants overnight at 4 °C. After pull-down with GSH-bead for 4 h at RT, pulled-down proteins were checked by IB. GAPDH was used as an internal loading control in all IB experiments. Error bars represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 is represented as asterisk (*), otherwise non-significant (ns).
XIAP and USP7 colocalize and physically interact A Representative image of immunofluorescence staining of USP7 (green), XIAP (red) in HeLa, HCT116, and A549 cells. Images were captured at 60X optical magnification; Scale bar represents 20 µm. B Purified Myc-His-USP7 incubated separately with GST and GST-XIAP in the presence of GSH- beads for 4 h. SDS-PAGE separated samples were stained with Coomassie blue to visualize the specific bands for GST, GST-XIAP, and Myc-His USP7. C HEK293 cell lysate was incubated separately with purified GST and GST-XIAP in the presence of GSH-beads for 4 h, followed by IB using indicated antibodies. D HEK293 cell lysate was immunoprecipitated with USP7 antibody followed by IB using indicated antibodies. E Interaction in overexpressed conditions: HEK293 cells were transiently transfected with FLAG-USP7 and GFP-XIAP or GFP as the control, followed by immunoprecipitation with anti-Flag antibody, and IB was performed with antibodies against Flag and GFP. F HEK293 cells were treated with either DMSO or MG132 (25 µM) for 8 h. Equal amounts of lysates were used for IP using USP7 antibody followed by IB with indicated antibodies. Veriblot secondary antibody was used to prevent nonspecific detection of the heavy and light chains. G Venn diagram shows a comparison of top-six interacting domain pairs of USP7 and XIAP obtained using three different docking software. H Upper panels showing full-length USP7 and XIAP proteins generated by stitching the available USP7 and XIAP domains in PDB. Lower panels were showing USP7-XIAP complex, where interface region was highlighted in yellow. I Schematic representation of USP7 deletion mutants interacting with full-length XIAP. J HEK293 cells were co-transfected with indicated plasmids; cell lysates were pull-down with Ni-NTA beads for 1 hr at room temperature. Pulled-downed and input were analyzed by IB with the indicated antibodies. Ponceau S staining indicates equal loading. K Schematic representation of XIAP deletion mutants interacting with full-length USP7. L HEK293 cells were transiently transfected with Flag-USP7. The lysates were incubated separately with GST or GST-XIAP deletion mutants overnight at 4 °C. After pull-down with GSH-bead for 4 h at RT, pulled-down proteins were checked by IB. GAPDH was used as an internal loading control in all IB experiments. Error bars represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.05 is represented as asterisk (*), otherwise non-significant (ns).
… 
USP7 protects XIAP from ubiquitination-dependent degradation in p53 independent manner A–D HEK293 cells were co-transfected with indicated plasmids, 24 h post-transfection cells were treated with USP7 inhibitor P5091 for another 24 h (B). 4 h before harvest before cells were treated with MG132 (25 µM). Endogenous (C) or GFP tagged XIAP (A, B, D) were pulled down with indicated antibodies. Bead-bound proteins, along with 5% input, were analyzed by IB. E Cell lysates prepared from p53Wt and p53Null HCT116 cells subjected to IB with the indicated antibodies. RT-PCR was performed to detect the mRNA expression of indicated genes. F HCT116, and HEK293 cells were treated with Nutlin3A (5 µM) for 24 h, and the protein level of XIAP, p53, p21, and USP7 were analyzed by IB. The change in mRNA expression of mentioned proteins was detected by RT-PCR after being treated with 5 µM of Nutlin 3 A for 24 h in HCT116 p53 WT cells. G HCT116 (p53wt and p53Null) cells were treated with the indicated dose of Doxorubicin for 6 h. The mRNA and protein expression of indicated genes were detected by RT-PCR and IB.
USP7 protects XIAP from ubiquitination-dependent degradation in p53 independent manner A–D HEK293 cells were co-transfected with indicated plasmids, 24 h post-transfection cells were treated with USP7 inhibitor P5091 for another 24 h (B). 4 h before harvest before cells were treated with MG132 (25 µM). Endogenous (C) or GFP tagged XIAP (A, B, D) were pulled down with indicated antibodies. Bead-bound proteins, along with 5% input, were analyzed by IB. E Cell lysates prepared from p53Wt and p53Null HCT116 cells subjected to IB with the indicated antibodies. RT-PCR was performed to detect the mRNA expression of indicated genes. F HCT116, and HEK293 cells were treated with Nutlin3A (5 µM) for 24 h, and the protein level of XIAP, p53, p21, and USP7 were analyzed by IB. The change in mRNA expression of mentioned proteins was detected by RT-PCR after being treated with 5 µM of Nutlin 3 A for 24 h in HCT116 p53 WT cells. G HCT116 (p53wt and p53Null) cells were treated with the indicated dose of Doxorubicin for 6 h. The mRNA and protein expression of indicated genes were detected by RT-PCR and IB.
… 
USP7 regulates cellular fate through involvement of XIAP A HEK 293 cells were transfected as indicated, for 24 h followed by Dox treatment (0, 1, 2, 3 µM) for another 6 h, and the expression of the indicated proteins was analyzed by IB. The normalized value of XIAP was mentioned. The change in Clv caspase3 and Clv PARP was plotted in the representative graph. B HCT116 (p53Null) cells were transfected with indicated plasmids for 24 h, followed by Dox (1 µM) treatment for another 6 h. the expression of indicated proteins was analyzed by IB to detect apoptosis intensity. C Annexin V/PI apoptosis of HCT116 p53Null cells transfected as indicated. Before analysis, transfected cells were treated with 1 µM and 5 µM of DOX for another 12 h as indicated; per cent of cells at different phases as indicated in the bar graph. The normal and necrotic cell was detected in HCT116 p53Null cells in a similar USP7 overexpression and Dox concentration but for 24 h. per cent of normal and the necrotic cell populations represented as bar graph. D HCT116 (p53Null) cells were transfected with Flag-USP7, as mentioned. After 24 h, cells were treated with P5091 (10 µM for 24 h) or Doxorubicin (1 µM for 6 h). Cell lysates were analyzed by IB using the indicated antibodies.
+3
USP7 regulates cellular fate through involvement of XIAP A HEK 293 cells were transfected as indicated, for 24 h followed by Dox treatment (0, 1, 2, 3 µM) for another 6 h, and the expression of the indicated proteins was analyzed by IB. The normalized value of XIAP was mentioned. The change in Clv caspase3 and Clv PARP was plotted in the representative graph. B HCT116 (p53Null) cells were transfected with indicated plasmids for 24 h, followed by Dox (1 µM) treatment for another 6 h. the expression of indicated proteins was analyzed by IB to detect apoptosis intensity. C Annexin V/PI apoptosis of HCT116 p53Null cells transfected as indicated. Before analysis, transfected cells were treated with 1 µM and 5 µM of DOX for another 12 h as indicated; per cent of cells at different phases as indicated in the bar graph. The normal and necrotic cell was detected in HCT116 p53Null cells in a similar USP7 overexpression and Dox concentration but for 24 h. per cent of normal and the necrotic cell populations represented as bar graph. D HCT116 (p53Null) cells were transfected with Flag-USP7, as mentioned. After 24 h, cells were treated with P5091 (10 µM for 24 h) or Doxorubicin (1 µM for 6 h). Cell lysates were analyzed by IB using the indicated antibodies.
… 
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ARTICLE
USP7 targets XIAP for cancer progression: Establishment of a
p53-independent therapeutic avenue for glioma
Gouranga Saha
1
, Sibani Sarkar
1
, Partha S. Mohanta
1
, Krishna Kumar
2
, Saikat Chakrabarti
2
, Malini Basu
3
and Mrinal K. Ghosh
1
✉
© The Author(s), under exclusive licence to Springer Nature Limited 2022
Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein
substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon
USP7 inhibition is well established in cancer containing wild type p53, which operates through the ‘USP7-Mdm2-p53’axis. However,
in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have
identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a
potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7
inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed
that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and
XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7
correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a
novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting
apoptosis.
Oncogene (2022) 41:5061–5075; https://doi.org/10.1038/s41388-022-02486-5
INTRODUCTION
The ubiquitin-proteasome system is a multi-component protein
destruction machinery found in all eukaryotic cells. Besides the
protein-Ub targeting and degradation, the UPS system was found to
regulate multiple essential biological processes, including protein-
protein interaction, subcellular localization, and activation/deactiva-
tion of functional proteins [1,2]. A crucial component of the UPS
system is Deubiquitinases (DUB), which is a special type of enzyme
that has the potential to remove and modify the Ub tag from its
target molecule [3]. The importance of these enzymes lies in the fact
that these are critical factors in maintaining the balance in overall
cellular signalling. Hence, mutations or aberrant expression of the
DUBs result in several diseases, including cancer. The role of DUBs in
cancer is elaborate, from regulation of cell cycle, DNA damage
repair, chromatin remodeling, changes in signalling, epithelial to
mesenchymal transition (EMT) [1–3], cell migration, and apoptosis.
The Ubiquitin Specific Protease 7 (USP7/HAUSP) is a deubiquitinase
that modifies the length of poly-ubiquitin chains on its target
proteins and has been shown to fulfil different roles in various
biological processes ranging from viral infections to malignant
transformation [4]. The well-known substrate of USP7 is the p53-
MDM2 complex, and several studies have demonstrated that the
induction of apoptosis upon USP7 functional inhibition is primarily
due to the restoration of p53 [5]. Interestingly, while some studies
demonstrated a strict dependency on p53 restoration for USP7
inhibition induced apoptosis, others have suggested that the
inhibitionofUSP7causescelldeathevenintheabsenceof
functional p53, possibly through deregulation of other essential
cellular pathways regulated by USP7 [6,7]. The pathways that
regulate p53-independent apoptosis upon USP7 inhibition are
highly context-dependent and not universally applied. Hence,
additional exploration of the possible alternate route and mechan-
istic insights into USP7-mediated oncogenesis is essential.
The Inhibitor of apoptosis proteins (IAPs) functions as negative
regulators of caspases found to be frequently overexpressed in
various human cancers, thereby contributing to tumor survival,
progression and chemoresistance, hence found to be widely
targeted in many anticancer therapies [8]. XIAP is one of the well-
characterized members of the IAP group and is argued to be the
most potent IAP in terms of anti-apoptotic ability. XIAP contains
three baculovirus IAP repeat (BIR) domains, a ubiquitin association
(UBA) domain, and a RING E3 ligase domain used in substrate
recognition and ubiquitination of target proteins [9]. XIAP has
exhibited aberrant expression patterns throughout a broad
spectrum of human cancers, and its overexpression has been
linked to chemoresistance and overall poor prognosis in specific
patient sub-groups [10–14].
Here, we successfully identified the anti-apoptotic protein XIAP
as a possible candidate for controlling p53-independent apoptosis
under the influence of USP7’s deubiquitinase activity. In addition,
Received: 6 May 2022 Revised: 18 September 2022 Accepted: 23 September 2022
Published online: 15 October 2022
1
Cancer Biology & Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), TRUE Campus, CN-6, Sector–V, Salt
Lake, Kolkata- 700091 & 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India.
2
Structural Biology & Bioinformatics Division, CSIR-IICB, TRUE Campus, CN-6, Sector–V, Salt Lake,
Kolkata 700091, India.
3
Department of Microbiology, Dhruba Chand Halder College, South 24 Paraganas, PIN -743372 Dakshin Barasat, West Bengal, India.
✉email: mrinal.res@gmail.com
www.nature.com/onc
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... Previously, we revealed that USP7 interacts with WDR1 to prevent ubiquitination-mediated degradation of β-Catenin in PDAC [22]. Recently, accumulated studies documented diverse oncogenic roles of USP7 in human cancers, such as lung cancer [23,24], gastric cancer [25], glioma [26], and breast cancer [27]. At present, a variety of small molecular inhibitors, natural compounds and small molecular peptides against USP7 have shown specific USP7 inhibition and certain anti-tumor activity [20], such as USP7 inhibitor P5091 [28], which can reduce Wnt signal pathway activity by enhancing ubiquitin-mediated degradation of β-catenin. ...
... USP7 plays multifaceted roles in cancer biology, including its impact on cell proliferation, invasiveness, immune evasion, and stem cell maintenance [15,23,26,40], underscoring its significance as a potential therapeutic target for various cancer types. In this study, we further broaden the knowledge of USP7 in the Warburg effect of PDAC cells and decipher c-Myc as a direct target of USP7 to regulate the glycolytic metabolism (Fig. 7B). ...
Ubiquitin-specific protease 7 maintains c-Myc stability to support pancreatic cancer glycolysis and tumor growth
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Full-text available
  • Dec 2024
  • J TRANSL MED
Background The typical pathological feature of pancreatic ductal adenocarcinoma (PDAC) is a significant increase in stromal reaction, leading to a hypoxic and poorly vascularized tumor microenvironment. Tumor cells undergo metabolic reprogramming, such as the Warburg effect, yet the underlying mechanisms are not fully understood. Methods Interference and overexpression experiments were conducted to analyze the in vivo and in vitro effects of USP7 on the growth and glycolysis of tumor cells. Small-molecule inhibitors of USP7 and transgenic mouse models of PDAC were employed to assess the consequences of targeting USP7 in PDAC. The molecular mechanism underlying USP7-induced c-Myc stabilization was determined by RNA sequencing, co-IP and western blot analyses. Results USP7 is abnormally overexpressed in PDAC and predicts a poor prognosis. Hypoxia and extracellular matrix stiffness can induce USP7 expression in PDAC cells. Genetic silencing of USP7 inhibits the glycolytic phenotypes in PDAC cells, while its overexpression has the opposite effect, as demonstrated by glucose uptake, lactate production, and extracellular acidification rate. Importantly, USP7 promotes PDAC tumor growth in a glycolysis-dependent manner. The small-molecule inhibitor P5091 targeting USP7 effectively suppresses the Warburg effect and cell growth in PDAC. In a transgenic mouse model of PDAC, named KPC, P5091 effectively blocks tumor progression. Mechanistically, USP7 interacts with c-Myc, enhancing its stability and expression, which in turn upregulates expression of glycolysis-related genes. Conclusions This study sheds light on the molecular mechanisms underlying the Warburg effect in PDAC and unveils USP7 as a potential therapeutic target for improving PDAC treatment.
View
... pGZ21dx-GFP-PTEN-WT (GFP-PTEN), pIRES-CHIP, pIRES-CHIP-H260Q, pIRES-CHIP-K30A, pGZ21dx-GFP-XIAP and FLAG-USP7 plasmids were described previously (Ahmed et al., 2012;Saha et al., 2022). pRK5-HA-Ubiquitin-WT (#17608), pRK5-HA-Ubiquitin-KØ (#17603), pRK5-HA-Ubiquitin-K48R (#17604), pCI-HA-Nedd4-1 (#27002) and HA-NLS-PTEN (#10933) were procured from Addgene. ...
... Co-immunoprecipitation was performed in HEK293 cells as described previously (Saha et al., 2022). Briefly, cells were lysed using IP lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 10% glycerol and Protease Inhibitor Cocktail (Takara)]. ...
View
... USP7 overexpression occurs across all major breast cancer subtypes, including ER-positive, HER2-positive, and TNBC tumors, and is independent of TP53 mutation status. Thus, in line with recent reports, USP7 likely contributes to tumorigenesis through p53-independent mechanisms (Yi et al. 2023;Saha et al. 2022;Tavana et al. 2018). This broad expression pattern, coupled with the association between high USP7 (which was not certified by peer review) is the author/funder. ...
USP7 inhibition perturbs proteostasis and tumorigenesis in triple negative breast cancer
Preprint
Full-text available
  • Jan 2025
The deubiquitinase USP7 is a critical regulator of tumorigenesis, known for stabilizing the MDM2-p53 pathway. Emerging evidence highlights USP7's p53-independent roles in proliferation and tumorigenesis. Triple negative breast cancers frequently inactivate p53 and this disease subtype remains difficult to treat and in need of new therapeutic options. Our study reveals that USP7 is upregulated in TNBC patient tumors. Importantly, genetic and pharmacologic USP7 inactivation impaired tumor progression in TNBC models. To explore USP7’s role in p53-mutant TNBCs, we performed deep quantitative proteomics across TNBC cell lines, identifying shared USP7 targets involved in cell proliferation, genome stability, and proteostasis. Acute USP7 inactivation allowed us to infer proximally controlled proteins which are likely direct targets. Surprisingly, many of the proteins downregulated by USP7 inhibition are E3 ubiquitin ligases. Thus, a key USP7 function in TNBC is to antagonize the degradation of ubiquitinating enzymes, since these enzymes are often susceptible to auto-ubiquitination and degradation. Notably, we identified TOPORS, a dual ubiquitin- and SUMO-ligase, among novel USP7 substrates. TOPORS interacts with the BRCA1-A DNA damage repair complex suggesting a USP7-TOPORS-BRAC1-A axis that might further explain the continued proliferation of genomically unstable TNBCs. Collectively, these data nominate USP7 as a potential therapeutic in TNBC.
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... XIAP is a potent and versatile suppressor of apoptosis that directly inhibits specific members of the caspase family of cysteine proteases 11,34,35 . In addition to caspase inhibition, XIAP is also found to be aberrantly elevated in numerous human cancer types, such as bladder cancer 16 , renal cell carcinoma 36 , Colorectal cancer 37 , glioma 38 , lung adenocarcinoma 39 , and melanoma 40 . However, the role that XIAP played in GC progression remains unclear and still needs to be further explored. ...
LINC02139 interacts with and stabilizes XIAP to regulate cell proliferation and apoptosis in gastric cancer
Article
Full-text available
  • Nov 2024
Previous reports showed that long non-coding RNA (lncRNA) participates in the development and progression of tumors. Nevertheless, the effect of LINC02139 and its mechanism on gastric cancer (GC) is still unknown. We revealed that LINC02139 is upregulated in GC cell lines and tissues and high LINC02139 expression was correlated with the advancement of GC in patients. Functionally, overexpression of LINC02139 promoted, while knockdown of LINC02139 impaired GC cell proliferation, migration, and invasion in vitro and impeded tumorigenesis in a tumor xenograft model in vivo. Mechanistically, LINC02139 directly bound to XIAP and increased the protein level by maintaining its protein stability through inhibition of the ubiquitination and proteasome-dependent degradation pathway. Importantly, the regulatory function of XIAP in LINC02139-mediated oncogenic effects was demonstrated. Both in vitro and in vivo experiments showed that LINC02139 and XIAP collaboratively modulate GC cell growth and apoptosis. Analysis of clinical GC tissues further confirmed the upregulation of XIAP and the positive association between LINC02139 and XIAP expression. These findings established LINC02139 as a driver of tumorigenesis and highlighted the crucial involvement of the LINC02139-XIAP axis in GC progression, suggesting its potential as a promising therapeutic target for combating GC advancement.
View
... Therapeutic benefits in diseases such as cancers caused by inappropriate inhibition of cell death, may result from reinduction or triggering of apoptosis (53). Therefore, XIAP, as a potent inhibitor of cell death, may be involved in chemotherapy resistance and tumor aggressiveness in several cancers (45,52,54,55). High levels of XIAP may be a potential therapeutic target (18). ...
MicroRNA‑24 alleviates colorectal cancer progression via a rs28382740 single nucleotide polymorphism in the long noncoding region of X‑linked inhibitor of apoptosis protein
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Full-text available
  • Oct 2024
Colorectal cancer (CRC) is one of the most prevalent malignant diseases worldwide. Recurrence is associated with the poor survival of patients with CRC. Targeted therapy and precision medicine for recurrent CRC may improve the clinical outcome. Therefore, finding biomarkers that can detect CRC early, assess its prognosis and survival, and predict its treatment response is key to improving the clinical prognosis. The aim of this study was to assess CRC recurrence by analyzing molecular differences using postoperative specimens. Whole-exome sequencing was first used to evaluate the molecular differences in CRC tissues from patients with recurrent disease, and the results were then verified with tissue array methods. The regulation of single nucleotide polymorphisms (SNPs) in long noncoding regions of interest was analyzed in the presence of target microRNAs (miRs) using luciferase assays. The results demonstrated that in patients with recurrent CRC, the G allele was mainly detected at the rs28382740 SNP in the 3′-untranslated region of the X-linked inhibitor of apoptosis (XIAP)-encoding gene. From the tissue arrays, 60% (3/5) of patients with the G allele of the rs28382740 SNP were diagnosed with CRC recurrence, whilst only 10% (1/10) of patients without the G allele had recurrent CRC (P=0.077). Furthermore, XIAP levels were high in non-CRC (50%; 2/4) and CRC (75%; 3/4) tissues of patients with recurrent disease and CRC (54.5%; 6/11) tissues of patients without recurrent disease. However, but only 9.1% (1/11) of non-CRC tissues of nonrecurrent patients had significantly high XIAP expression levels (P=0.022). Using a luciferase assay, it was demonstrated that miR-24s (miR-24-1-5p and miR-24-2-5p) targeting the rs28382740 SNP reduced XIAP levels in CRC cells with rs28382740 SNP genotype G. These results indicate that apoptosis-related proteins, such as XIAP, may be therapeutic targets or biomarkers for tumor development. The data from the present study support an inhibitory effect of miR-24s on XIAP expression. However, this inhibitory potency depends on the rs28382740 SNP genotype and may alleviate CRC progression by regulating the expression of XIAP.
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... 23,24 For instance, USP7 deubiquitinates XIAP to enhance protein stability, thereby inhibiting cancer cell apoptosis. 28 USP7 is able to enhance cancer cell invasiveness and proliferation by stabilizing EZH2, depending on its deubiquitination. 29 USP7 has emerged as a promising target for anti-cancer therapy. ...
Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma
Article
Full-text available
  • Jul 2024
  • EUR J HISTOCHEM
Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the thyroid. Fibroblast growth factor receptor 1 (FGFR1) is highly expressed in PTC and works as an oncogenic protein in this disease. In this report, we wanted to uncover a new mechanism that drives overexpression of FGFR1 in PTC. Analysis of FGFR1 expression in clinical specimens and PTC cells revealed that FGFR1 expression was enhanced in PTC. Using siRNA/shRNA silencing experiments, we found that FGFR1 downregulation impeded PTC cell growth, invasion, and migration and promoted apoptosis in vitro, as well as suppressed tumor growth in vivo. Bioinformatic analyses predicted the potential USP7-FGFR1 interplay and the potential binding between YY1 and the FGFR1 promoter. The mechanism study found that USP7 stabilized FGFR1 protein via deubiquitination, and YY1 could promote the transcription of FGFR1. Our rescue experiments showed that FGFR1 re-expression had a counteracting effect on USP7 downregulation-imposed in vitro alterations of cell functions and in vivo suppression of xenograft growth. In conclusion, our study identifies the deubiquitinating enzyme USP7 and the oncogenic transcription factor YY1 as potent inducers of FGFR1 overexpression. Designing inhibitors targeting FGFR1 or its upstream inducers USP7 and YY1 may be foreseen as a promising strategy to control PTC development.
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... Ubiquitin-specific protease 7 (USP7) is an extensively researched deubiquitination enzyme (DUB) [33]. Its enzymatic activity influences the localization, activation, and stability of its substrates [34,35]. ...
The deubiquitinase USP7 and E3 ligase TRIM21 regulate vasculogenic mimicry and malignant progression of RMS by balancing SNAI2 homeostasis
Article
Full-text available
  • May 2024
  • J EXP CLIN CANC RES
Background Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported. Methods Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2. Results Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS. Conclusion Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03056-1.
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Emerging insights into HAUSP (USP7) in physiology, cancer and other diseases
Article
Full-text available
  • Jun 2018
Herpesvirus-associated ubiquitin-specific protease (HAUSP) is a USP family deubiquitinase. HAUSP is a protein of immense biological importance as it is involved in several cellular processes, including host-virus interactions, oncogenesis and tumor suppression, DNA damage and repair processes, DNA dynamics and epigenetic modulations, regulation of gene expression and protein function, spatio-temporal distribution, and immune functions. Since its discovery in the late 1990s as a protein interacting with a herpes virus regulatory protein, extensive studies have assessed its complex roles in p53-MDM2-related networks, identified numerous additional interacting partners, and elucidated the different roles of HAUSP in the context of cancer, development, and metabolic and neurological pathologies. Recent analyses have provided new insights into its biochemical and functional dynamics. In this review, we provide a comprehensive account of our current knowledge about emerging insights into HAUSP in physiology and diseases, which shed light on fundamental biological questions and promise to provide a potential target for therapeutic intervention.
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Targeting USP7-Mediated Deubiquitination of MDM2/MDMX-p53 Pathway for Cancer Therapy: Are We There Yet?
Article
Full-text available
  • Apr 2020
The p53 tumor suppressor protein and its major negative regulators MDM2 and MDMX oncoproteins form the MDM2/MDMX-p53 circuitry, which plays critical roles in regulating cancer cell growth, proliferation, cell cycle progression, apoptosis, senescence, angiogenesis, and immune response. Recent studies have shown that the stabilities of p53, MDM2, and MDMX are tightly controlled by the ubiquitin-proteasome system. Ubiquitin specific protease 7 (USP7), one of the most studied deubiquitinating enzymes plays a crucial role in protecting MDM2 and MDMX from ubiquitination-mediated proteasomal degradation. USP7 is overexpressed in human cancers and contributes to cancer initiation and progression. USP7 inhibition promotes the degradation of MDM2 and MDMX, activates the p53 signaling, and causes cell cycle arrest and apoptosis, making USP7 a potential target for cancer therapy. Several small-molecule inhibitors of USP7 have been developed and shown promising efficacy in preclinical settings. In the present review, we focus on recent advances in the understanding of the USP7-MDM2/MDMX-p53 network in human cancers as well as the discovery and development of USP7 inhibitors for cancer therapy.
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Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism
Article
Full-text available
  • Mar 2020
Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.
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Promyelocytic Leukemia (PML) gene regulation: implication towards curbing oncogenesis
Article
Full-text available
  • Sep 2019
Dysregulation of PML, a significant tumor suppressor is linked with cancers of different histological origins, with a decreased expression observed with a higher tumor grade. This necessitates studying the mechanisms to maintain a stable expression of PML. However much less is known about the transcriptional regulation of PML, more so in the context of breast carcinoma. ERβ has emerged as a critical factor in understanding breast cancer, especially since a huge proportion of breast cancers are ERα− and thus insensitive to tamoxifen therapy. This study aims to uncover an unidentified mechanism of PML gene regulation and its stabilization in breast cancer via ERβ signalling and the impact on cellular apoptosis. We found that clinical expression of PML positively correlates with that of ERβ both in normal and breast carcinoma samples and inversely correlates with markers of cellular proliferation, hinting towards a possible mechanistic interdependence. Both mRNA and protein expression of PML were increased in response to ERβ overexpression on multiple human breast cancer cell lines. Mechanistically, luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that ERβ can interact with the PML promoter via ERE and AP1 sites to enhance its transcription. ERβ induced stable PML expression causes a decline of its target protein Survivin and simultaneously provides a stable docking platform leading to stabilisation of its target Foxo3a, further causing transcriptional upregulation of pro-apoptotic factors p21 and p27. Immunohistochemical analyses of cancer and normal breast tissues and functional assays conducted corroborated the findings. Collectively, our study identifies ERβ signalling as a novel mechanism for PML gene regulation in ERα− breast cancer. It also reveals bi-directional downstream effect in which ‘ERβ-PML-(Foxo3a/Survivin)’ network acts as a therapeutic axis by suppressing cellular survival and promoting cellular apoptosis in breast carcinoma.
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Identification of pharmacodynamic biomarkers and common molecular mechanisms of response to genotoxic agents in cancer cell lines
Article
Full-text available
  • Oct 2019
  • CANCER CHEMOTH PHARM
Purpose Genotoxic agents (GAs) including cisplatin, doxorubicin, gemcitabine, and topotecan are often used in cancer treatment. However, the response to GAs is variable among patients and predictive biomarkers are inadequate to select patients for treatment. Accurate and rapid pharmacodynamics measures of response can, thus, be useful for monitoring therapy and improve clinical outcomes. Methods This study focuses on integrating a database of genome-wide response to treatment (The NCI Transcriptional Pharmacodynamics Workbench) with a database of baseline gene expression (GSE32474) for the NCI-60 cell lines to identify mechanisms of response and pharmacodynamic (PD) biomarkers. Results and conclusions Our analysis suggests that GA-induced endoplasmic reticulum (ER) stress may signal for GA-induced cell death. Reducing the uptake of GA, activating DNA repair, and blocking ER-stress induction cooperate to prevent GA-induced cell death in the GA-resistant cells. ATF3, DDIT3, CARS, and PPP1R15A appear as possible candidate PD biomarkers for monitoring the progress of GA treatment. Further validation studies on the proposed intrinsic drug-resistant mechanism and candidate genes are needed using in vivo data from either patient-derived xenograft models or clinical chemotherapy trials.
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RNA helicase p68 deploys β-catenin in regulating RelA/p65 gene expression: implications in colon cancer
Article
Full-text available
  • Dec 2019
  • J EXP CLIN CANC RES
Background: RelA/p65 a crucial member of NF-κB signaling pathway plays diverse role in mediating oncogenesis. Limited knowledge prevails on the mechanistic insights of RelA gene regulation. RNA helicase p68 apart from being a vital player of RNA metabolism acts as a transcriptional coactivator of several oncogenic transcription factors including β-catenin and is highly implicated in cancer progression. In this study, we aim to discern the molecular mechanism of how an RNA helicase, p68 deploys a major oncogenic signaling pathway, Wnt/ β-catenin to regulate the expression of RelA, an indispensable component of NF-κB signaling pathway towards driving colon carcinogenesis. Methods: Immunoblotting and quantitative RT-PCR was performed for determining the protein and mRNA expressions of the concerned genes respectively. Luciferase assay was employed for studying the promoter activity of RelA. Chromatin immunoprecipitation was used to evaluate the occupancy of transcription factors on the RelA promoter. Immunohistochemical analysis was conducted using FFPE sections derived from normal human colon and colon cancer patient samples. Finally, a syngeneic colorectal allograft mouse model was used to assess physiological significance of the in vitro findings. Results: p68, β-catenin and RelA proteins were found to bear strong positive correlation in normal and colon carcinoma patient samples. Both p68 and β-catenin increased RelA mRNA and protein expression. p68, β-catenin and Wnt3a elevated RelA promoter activity. Conversely, p68 and β-catenin knockdown diminished RelA promoter activity and led to reduced RelA mRNA and protein expression. p68 was perceived to occupy RelA promoter with β-catenin at the TCF4/LEF (TBE) sites thereby potentiating RelA transcription. p68 and β-catenin alliance positively modulated the expression of signature NF-κB target genes. Enhanced NF-κB target gene expression by p68 was corroborated by findings in clinical samples. Tumors generated in mice colorectal allograft model, stably expressing p68 further reinforced our in vitro findings. Conclusions: We report for the first time a novel mechanism of alliance between p68 and β-catenin in regulating the expression of RelA and stimulating the NF-κB signaling axis towards driving colon carcinogenesis. This study unravels novel modes of p68-mediated colon carcinogenesis, marking it a potential target for therapy.
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The p53 Pathway in Glioblastoma
Article
Full-text available
  • Sep 2018
The tumor suppressor and transcription factor p53 plays critical roles in tumor prevention by orchestrating a wide variety of cellular responses, including damaged cell apoptosis, maintenance of genomic stability, inhibition of angiogenesis, and regulation of cell metabolism and tumor microenvironment. TP53 is one of the most commonly deregulated genes in cancer. The p53-ARF-MDM2 pathway is deregulated in 84% of glioblastoma (GBM) patients and 94% of GBM cell lines. Deregulated p53 pathway components have been implicated in GBM cell invasion, migration, proliferation, evasion of apoptosis, and cancer cell stemness. These pathway components are also regulated by various microRNAs and long non-coding RNAs. TP53 mutations in GBM are mostly point mutations that lead to a high expression of a gain of function (GOF) oncogenic variants of the p53 protein. These relatively understudied GOF p53 mutants promote GBM malignancy, possibly by acting as transcription factors on a set of genes other than those regulated by wild type p53. Their expression correlates with worse prognosis, highlighting their potential importance as markers and targets for GBM therapy. Understanding mutant p53 functions led to the development of novel approaches to restore p53 activity or promote mutant p53 degradation for future GBM therapies.
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Proteogenomic and metabolomic characterization of human glioblastoma
Article
  • Feb 2021
  • CANCER CELL
Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.
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Negative feedback regulation of wild-type p53 biosynthesis.
Article
  • Sep 1995
When growth‐arrested mouse fibroblasts re‐entered the cell‐cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma‐irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed‐back loop: p53 mRNA has a predicted tendency to form a stable stem‐loop structure that involves the 5′‐untranslated region (5′‐UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5′‐UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53‐intrinsic RNA re‐annealing activity. The inhibition of p53 biosynthesis requires wild‐type p53, as it is not observed with MethA mutant p53, p53‐catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem‐loop structures. Release from negative feed‐back regulation of p53 biosynthesis, e.g. after damage‐induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell‐cycle checkpoint determinant after DNA damage.
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Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis
Article
  • Sep 2018
Central to the recognition, signaling, and repair of DNA double-strand breaks (DSBs) are the MRE11-RAD50-NBS1 (MRN) complex and mediator of DNA damage checkpoint protein 1 (MDC1), the interplay of which is essential for initiation and amplification of the DNA damage response (DDR). The intrinsic rule governing the regulation of the function of this molecular machinery remains to be investigated. We report here that the ubiquitin-specific protease USP7 was physically associated with the MRN-MDC1 complex and that the MRN-MDC1 complex acted as a platform for USP7 to efficiently deubiquitinate and stabilize MDC1, thereby sustaining the DDR. Accordingly, depletion of USP7 impaired the engagement of the MRN-MDC1 complex and the consequent recruitment of the downstream factors p53-binding protein 1 (53BP1) and breast cancer protein 1 (BRCA1) at DNA lesions. Significantly, USP7 was overexpressed in cervical cancer, and the level of its expression positively correlated with that of MDC1 and worse survival rates for patients with cervical cancer. We demonstrate that USP7-mediated MDC1 stabilization promoted cervical cancer cell survival and conferred cellular resistance to genotoxic insults. Together, our study reveals a role for USP7 in regulating the function of the MRN-MDC1 complex and activity of the DDR, supporting the pursuit of USP7 as a potential therapeutic target for MDC1-proficient cancers.
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