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, 22 (10), 1180-1186

HAUSP Deubiquitinates and Stabilizes N-Myc in Neuroblastoma

Omid TavanaDawei Li ... Wei Gu

HAUSP Deubiquitinates and Stabilizes N-Myc in Neuroblastoma

Omid Tavana et al. Nat Med.

Abstract

The MYCN proto-oncogene is amplified in a number of advanced-stage human tumors, such as neuroblastomas. Similar to other members of the MYC family of oncoproteins, MYCN (also known as N-Myc) is a transcription factor, and its stability and activity are tightly controlled by ubiquitination-dependent proteasome degradation. Although numerous studies have demonstrated that N-Myc is a driver of neuroblastoma tumorigenesis, therapies that directly suppress N-Myc activity in human tumors are limited. Here we have identified ubiquitin-specific protease 7 (USP7; also known as HAUSP) as a regulator of N-Myc function in neuroblastoma. HAUSP interacts with N-Myc, and HAUSP expression induces deubiquitination and subsequent stabilization of N-Myc. Conversely, RNA interference (RNAi)-mediated knockdown of USP7 in neuroblastoma cancer cell lines, or genetic ablation of Usp7 in the mouse brain, destabilizes N-Myc, which leads to inhibition of N-Myc function. Notably, HAUSP is more abundant in patients with neuroblastoma who have poorer prognosis, and HAUSP expression substantially correlates with N-Myc transcriptional activity. Furthermore, small-molecule inhibitors of HAUSP's deubiquitinase activity markedly suppress the growth of MYCN-amplified human neuroblastoma cell lines in xenograft mouse models. Taken together, our findings demonstrate a crucial role of HAUSP in regulating N-Myc function in vivo and suggest that HAUSP inhibition is a potential therapy for MYCN-amplified tumors.

Figures

Figure 1
Figure 1. HAUSP affects and directly interacts with N-Myc both in vitro and in vivo
(a) Western blot of mouse brains collected at day E13.5; n = 2. (b) Representative immunohistochemistry of HAUSP, N-Myc and p53 of E18.5 mouse cortex sections of the marginal zone to the cortical plate separated by a dotted black line; magnification 40 ×; scale bar 25 μm; n = 2 per group. (c) N-Myc expression vector cotransfected with Flag-HAUSP (lane 2) or empty vector (lane 1) in HEK293T cells. Cell lysates were incubated with Flag/M2 beads then subjected to western blot; 10% input; n = 3. (d, e) Endogenous immunoprecipitation of N-Myc with HAUSP from native IMR-32 (d) and SK-N-BE(2)C cells (e); 2% input; n = 3. (f) Direct interaction between purified Flag-N-Myc and GST-HAUSP. Purified N-Myc was incubated with GST protein (lane 1) or GST-HAUSP (lane 2) and immobilized with GST beads then subjected to western blot; 10% input; n = 2. (g) Direct interaction between purified Flag-HAUSP and GST-N-Myc as in Fig. 1f. Proteins were immobilized with GST beads then subjected to western blot; 1% input; n = 3. (h) Schematic representation of N-Myc deletion mutants used for domain mapping. (i) Indicated N-Myc expression vectors cotransfected with HAUSP in HEK293T cells. Lysates were incubated with HA beads then subjected to western blot; 10% input; n = 3.
Figure 2
Figure 2. HAUSP regulates N-Myc through deubiquitination
(a) Western blot of HEK293T cells transfected with N-Myc and empty vector, or increasing Flag-HAUSP; n = 3. (b) Western blot of HEK293T cells transfected with indicated HA-N-Myc constructs and empty vector (—) or HAUSP (+); n = 3. (c) Western blot of HEK293T cells transfected with N-Myc and empty vector, HAUSP wild-type (WT), or HAUSP C223S (CS); n = 3. (d) Western blot of HEK293T cells transfected with N-Myc and empty vector, Flag-HAUSP (WT) (left), or Flag-HAUSPcs (right). Cyclohexamide treatment (CHX, 50 ug/ml) for indicated times; n = 3. (e) Ubiquitin (Ub) and Flag-N-Myc expression vectors transfected with empty vector (lane 3) or in combination with HAUSP WT (lane 4) or HAUSP CS (lane 5) into HEK293T cells. Lanes 1—2 serve as controls. Lysates were incubated with Flag/M2 beads then subjected to western blot; n = 2. (f) Western blot of SK-N-DZ cells transfected with two-rounds of control RNAi, HAUSP pool, or individual HAUSP oligos composing the pool; n = 3. Western blot of LAN-1 (lanes 7—8) or SK-N-BE(2)C (lanes 9—10) cells transfected with three-rounds of control RNAi or HAUSP pool; n = 3. (g) Cell growth assay of SK-N-DZ expressing shGFP, HAUSP-shRNA #1, or HAUSP-shRNA #2. Error bars represent the mean ± SEM of 3 independent experiments. * represents a P value < 0.05 using two-tailed Student's t-test. (h) Colony formation assay using SK-N-DZ cells from Fig. 2g; scale bar 14.5 mm; n = 3. (i) Western blot of H1299 cells stably expressing an N-Myc polypeptide transfected with three-rounds of control RNAi or HAUSP pool; n = 3.
Figure 3
Figure 3. Pharmacologically blocking HAUSP deubiquitination in neuroblastoma cells
(a) Ubiquitin and Flag-N-Myc vectors transfected with empty vector (lane 2) or in combination with HAUSP (lane 3—4) into HEK293T cells. Flag-N-Myc was transfected with empty vector (lane 1). Cells were treated with 20 μm of P22077 for the last 8 hours (lane 4). Lanes 1—3 were treated with DMSO. Cell lysates were incubated with Flag/M2 beads then subjected to western blot; n = 3. (b) Western blot analysis of indicated neuroblastoma cells treated with 20 μm of DMSO (—) or P22077 (+) (upper) and 12.5 μm of P5091 (+) (bottom) for 8 hours; n = 4. (c) Western blot analysis of SK-N-DZ cells treated for 6 hours with DMSO (—) or 10 μm of P22077 (+) or additionally treated with MG132 (lanes 3—4); n = 3. (d—i) Cell growth assay of SK-N-DZ (d), IMR-5 (e), IMR-32 (f), LAN-1 (g), SK-N-AS (h) NB-16 (i) cells treated either with DMSO or 10 μm of P22077. Error bars represent the mean ± SEM of 3 independent experiments. * represents a P value < 0.05 using two-tailed Student's t-test. (j) Colony assay using indicated neuroblastoma cells singly treated with DMSO or 10 μm of P22077; scale bar 8 mm; n = 3.
Figure 4
Figure 4. The role of HAUSP on the pathogenesis of neuroblastomas
(a) Kaplan-Meier analyses for SEQC RNA-seq database (left) and Kocak database (right) of overall survival for only MYCN-amplified samples. (b) Correlation of USP7 expression versus N-Myc transcriptional activity in neuroblastoma patients; MYCN-amplification (red) MYCN non-amplification (grey). (c—e) Pre-clinical neuroblastoma mouse model (see methods) (c) Representative in vivo bioluminescence imaging of tumor-bearing mice from day 12. Luciferase activity: low = blue, high = red; scale bar 13 mm. (d; top) Representative images of tumor-bearing kidneys versus normal kidney at day 15; scale bar 10 mm. (d; bottom) Tumor weight from day 15; n = 8 mice per group; ** represents a P value < 0.005 using two-tailed Student's t-test. (e) Representative immunohistochemistry of SK-N-DZ tumors for HAUSP, N-Myc, and Ki-67 from day 15. Magnification 40 ×; scale bar 25 μm; n = 3. (f—i) Subcutaneous xenograft models (see methods); (f) SK-N-DZ tumor image and weight; scale bar 10 mm; n = 5 mice per group; * represents a P value < 0.05 using two-tailed Student's t-test. (g) Representative immunohistochemistry of tumors harvested at day 12 stained for HAUSP, N-Myc, and Ki-67; Magnification 40 ×; scale bar 12.5 μm; n = 3. (h) NB-16 tumor image and weight; scale bar 10 mm; n = 5 mice per group. (i) Representative immunohistochemistry of tumors harvested at day 12 stained for HAUSP, N-Myc, and Ki-67; Magnification 40 ×; scale bar 12.5 μm; n = 3. **Note: Because NB-16 derived tumors grow faster in the subcutaneous model compared with SK-N-DZ, treatment started two weeks post implantation while with SK-N-DZ derived tumors, treatment began three weeks after implantation to more closely account for tumor size. Both SK-N-DZ and NB-16 subcutaneous models were treated with the same dose and time period (See Supplementary Fig. 6g).

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References

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