HELZ2 promotes K63-linked polyubiquitination of c-Myc to induce retinoblastoma tumorigenesis

Retinoblastoma is a rare ocular tumor in children that originates in the retina. Several core transcriptional regulators maintain the expansion of retinoblastoma tumors, including c-Myc. Here, we demonstrated that Helicase with zinc finger domain 2 (HELZ2) promoted retinoblastoma tumorigenesis by targeting c-Myc. HELZ2-deficient inhibited retinoblastoma cell proliferation, whereas overexpression of HELZ2 promoted retinoblastoma cell proliferation. In addition, high levels of HELZ2 promoted xenograft retinoblastoma tumorigenesis and inhibited animal survival. Mechanistically, HELZ2 interacted with c-Myc and promoted its K63-linked polyubiquitination. We indicated that HELZ2 promoted the interaction between E3 ubiquitin ligase HUWE1 and c-Myc, and HELZ2-mediated K63-linked polyubiquitination and activation of c-Myc were dependent on HUWE1. Taken together, HELZ2 plays a critical role in the regulation of retinoblastoma tumorigenesis by enhancing the activity of c-Myc.


Introduction
Retinoblastoma was first described by Pawius in the sixteenth century [1]. Retinoblastoma is the most common intraocular malignancy in children, accounting for 3% of all childhood cancers [2]. Tumor formation usually begins with mutation in both alleles of the retinoblastoma tumor suppressor gene RB1, followed by a series of other genetic alterations that correlate with the clinical stage and pathologic findings of the tumor [3]. The annual incidence rate of newborns worldwide is around 1 in 12,000 to 1 in 20,000, and the incidence rate of retinoblastoma continues to increase [4]. Meanwhile, the incidence is higher in underdeveloped countries, which are often accompanied by metastasis, recurrence, and poor prognosis [5]. According to the different conditions observed by the patient, retinoblastoma can be treated in many methods, such as enucleation, radiotherapy, chemotherapy, and cryotherapy [6]. Nevertheless, there are still many shortcomings in the treatment of high-risk retinoblastoma recurrence. Therefore, in-depth exploration of the intracellular signal transmission mechanism of retinoblastoma is essential to discover potential therapeutic targets. c-Myc oncoprotein is a transcription factor that can regulate a variety of genes, such as cell proliferation [7]. Proper c-Myc activity and stability are essential for controlled cell proliferation.
Ubiquitination-associated post-translational modification plays an important role in c-Myc protein activity and stability regulation. c-Myc can be ubiquitinated by a variety of E3 ubiquitin ligases, such as SCF FBXO28 , SCF b−TRCP , TRIM32, and SCF Fbw7 [8]. Depending on the specific E3 ligase, ubiquitination can positively or negatively regulate the activity and stability of c-Myc. HUWE1 is a member of the HECTdomain containing ubiquitin E3s that enhances c-Myc activity by promoting its K63-linked polyubiquitination [9,10]. Our previous studies have demonstrated that ZCCHC2 suppresses retinoblastoma tumorigenesis by inhibiting HUWE1mediated K63-linked polyubiquitination of c-Myc [11]. In this study, we demonstrate that HELZ2 positively regulates retinoblastoma cell proliferation and tumorigenesis. HELZ2 promotes c-Myc K63-linked polyubiquitination by promoting the interaction of HUWE1 with c-Myc.

Animals
Eight-to ten-week-old and age-matched BALB/c nude mice had free access to water and food in a temperaturecontrolled room (23-25 °C) and relative humidity of 40-70%, the light/dark cycle is 12 h, and there are 5 mice in each cage. Allow the animals to acclimate to the environment for at least 7 days before experiment. All animals were randomly and injected subcutaneously on both flanks with 1 × 10 6 Y79 and HXO-RB44 cells expressing the NC, gHELZ2, gHELZ2 + HELZ2. All animal use and experimental protocols were carried out in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines and approved by the Animal Care and Ethics Committee of Wuhan University of Medicine.

Genome-wide CRISPR/Cas9 knockout screen
Cas9 expressing cells are transduced using a mixed lentivirus guide library, targeting thousands of genes, each with several guide sequences. The cells then multiply for several weeks. Over time, the guide sequences that target adaptive genes that cause cell death will gradually run out, while the guide sequences that target growth-suppressing genes become more abundant. Then prepare genomic DNA (gDNA) from the samples (day 14 after transduction) and control samples (day 3 after transduction). The leader sequence is amplified by PCR, and the relative frequency of the leader in the two populations is determined by next-generation sequencing (NGS). Assess the enrichment or consumption of the guide sequence relative to the control. sgRNA, single guide RNA.

Cell viability
Cell viability was assessed by the ability of cells to absorb Thiazole Blue Tetrazolium Bromide (MTT) (Bio-Tek, USA).
The method is carried out in accordance with the manufacturer's instructions.

Immunoprecipitation and immunoblotting analysis
Cells were lysed in 1 ml NP-40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation reaction, incubate 0.4 ml of lysate with 0.5-2 μg of the designated antibody or control IgG and 35 μL of 1:1 protein-G Sepharose (GE Healthcare) slurry at 4 °C for 3 h. Sepharose beads use 1 ml of lysis buffer containing 500 mM NaCl wash 3 times [12]. The precipitates were fractionated by SDS-PAGE, and immunoblotting analysis was performed with the indicated antibodies. Antibodies used in this study were purchased from the following indicated companies: HA- Tag

Statistics
All data are expressed as the mean ± SE. Newman-Keuls tests were used for performing ad hoc comparisons when appropriate. Student's t-tests and variance analyses were used to determine differences among groups. p < 0.05 was considered statistically significant.

HELZ2 promotes retinoblastoma cell proliferation
To explore the potential therapeutic targets of retinoblastoma, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for retinoblastoma Y79 cell proliferation, and HELZ2 was identified as a candidate protein (Fig. 1a). Knockout of HEZL2 inhibited Y79 cell proliferation (Fig. 1b). Overexpression of HELZ2 promoted Y79 cell proliferation (Fig. 1c). In addition, we further determined the function of HELZ2 in retinoblastoma cell proliferation. We created HELZ2-deficient Y79 and HXO-RB44 cells (Fig. 1d, e). We found that HELZ2 deficiency inhibited retinoblastoma cell proliferation, while cell growth was restored after rescuing HELZ2 (Fig. 1d, e). Together, these results demonstrate that HELZ2 is potential positively regulator of retinoblastoma cell proliferation.

HELZ2 promotes retinoblastoma tumor development
We further subcutaneously injected the NC, gHELZ2, or gHELZ2+HELZ2 retinoblastoma cells into BALB/c nude mice, and showed that cells with HELZ2-deficient performed lower retinoblastoma growth and greater animal survival ( Fig. 2a-d). These results suggested that HELZ2 promotes retinoblastoma growth tumorigenesis.

HELZ2 promotes c-Myc K63-linked polyubiquitination
c-Myc is a well-known transcription factor that controls most actively transcribed genes and regulates many tumorigeneses [13,14]. To identify whether HELZ2-regulated cell proliferation is associated with c-Myc, we performed co-immunoprecipitation assays. Results showed that HELZ2 interacted with c-Myc (Fig. 3a). The focus of c-Myc research is its stability and activity. Immunoblot analysis of c-Myc protein levels in HELZ2-deficient Y79 and HXO-RB44 cells, and showed that c-Myc protein levels did not show significant difference after HELZ2deficient (Fig. 3b), meanwhile, the mRNA level of MYC also did not show significant difference (Fig. 3c). Therefore, we tested the c-Myc activity after overexpression of HELZ2. We transfected Flag-c-Myc and HA-Ub or its mutants (K48O or K63O, where all lysine residues were replaced by arginine except K48 or K63, respectively) with HELZ2, results showed that overexpression of HELZ2 promoted c-Myc K63-linked polyubiquitination (Fig. 3d). Meanwhile, endogenous HELZ2-deficient inhibited c-Myc K63-linked polyubiquitination (Fig. 3e). Taken together, our data indicated that HELZ2 promotes K63-linked polyubiquitination of c-Myc.

Discussion
In this study, we demonstrated that HELZ2 plays a critical role in c-Myc-associated retinoblastoma tumorigenesis. HELZ2 positively regulated retinoblastoma cell proliferation and tumor development. HELZ2 interacted with c-Myc and promoted its K63-linked polyubiquitination. We further indicated that HELZ2-mediated K63-linked polyubiquitination of c-Myc was dependent on HUWE1.
We performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for retinoblastoma cell proliferation. Our results identified that HELZ2 was a positive candidate protein for retinoblastoma cell proliferation. HELZ2-deficient inhibited retinoblastoma cell proliferation, whereas overexpression of HELZ2 promoted. Meanwhile, HELZ2-deficient inhibited subcutaneously retinoblastoma growth and promoted animal survival. Taken together, we indicated that HELZ2 promotes retinoblastoma cell proliferation and tumorigenesis.
In addition, we found that HELZ2 interacted with c-Myc, HELZ2-deficient did not affect c-Myc stability. However, overexpression of HELZ2 promoted c-Myc K63-linked polyubiquitination, while loss of HELZ2 inhibited. HUWE1 is a c-Myc ubiquitin E3 ligase that enhances c-Myc activity by promoting its K63-linked polyubiquitination. We found that HELZ2 was associated with HUWE1 and overexpression of HELZ2 promoted interaction of c-Myc with HUWE1, meanwhile, HELZ2-deficient had the opposite  Fig. 4 HELZ2 promotes HUWE1-mediated c-Myc K63-linked polyubiquitination. a HELZ2 interacts with HUWE1. HEK293T cells were transfected with indicated plasmids for 20 h followed by coimmunoprecipitation and immunoblotting analysis. b HELZ2 promotes the association of c-Myc with HUWE1. HEK293T cells were transfected with indicated plasmids for 20 h followed by co-immunoprecipitation and immunoblotting analysis. c HELZ2-deficient inhibits the association of c-Myc with HUWE1. HEK293T cells were transfected with indicated plasmids for 20 h followed by co-immunoprecipitation and immunoblotting analysis. d HELZ2-mediated polyubiquitination of c-Myc is dependent on HUWE1. Control or HUWE1-deficient HEK293T cells were transfected with indicated plasmids for 20 h followed by co-immunoprecipitation and immunoblotting analysis tumorigenesis. HELZ2 interacted with c-Myc and promoted HUWE1-mediated c-Myc K63-linked polyubiquitination. HELZ2 may be a potential therapeutic target for retinoblastoma.