SPOP suppresses prostate cancer through regulation of CYCLIN E1 stability

SPOP is one of the important subunits for CUL3/SPOP/RBX1 complex tightly connected with tumorigenesis. However, its exact roles in different cancers remain debatable. Here, we identify CYCLIN E1, as a novel substrate for SPOP. SPOP directly interacts with CYCLIN E1 and specific regulates its stability in prostate cancer cell lines. SPOP/CUL3/RBX1 complex regulates CYCLIN E1 stability through poly-ubiquitination. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting that SPOP probably regulates the stability of CDK2-free CYCLIN E1. CYCLIN E1 expression rescued proliferation, migration, and tumor formation of prostate cancer cell suppressed by SPOP. Furthermore, we found SPOP selectively regulates the substrates’ stability and signaling pathways in prostate cancer and CCRC cell lines, suggesting that complicated mechanisms exist for SPOP to regulate substrate specificity. Altogether, we have revealed a novel mechanism for SPOP in suppressing prostate cancer and provided evidence to show SPOP has dual functions in prostate cancer and CCRC.

HEK293 cells were transfected with indicated plasmids followed by treatment with 10 μM MG132 for 12 hr. Co-IP was performed to map the CYCLIN E1 domains interacting with SPOP. D. HEK293 cells were transfected with indicated plasmids for followed by treatment with 10 μM MG132 for 12 hr.  wild type and mutants T77A, T395A, T77/395A were transfected into DU145 with or without FBXW7β, and western blotting was carried out with the indicated antibodies.
GFP was co-transfected and blotted as control for transfection efficiency.

Protein expression in bacteria and GST purification
Individual cDNA sequences were cloned into pGEX-KG vector. The constructs were transformed into BL-21 bacteria, which were induced with 0.2 mM IPTG at 18℃ for 4 hr. The harvested cells were sonicated and the lysates were centrifuged at 10,000×g for 1h. Recombinant proteins were purified from the supernatant with Glutathione Sepharose 4 according to manufacturer's instructions (GE Healthcare). Protein concentration was quantified by Qubit 2.0 (Invitrogen).
The beads were spin down and washed three times with lysis buffer. After the final wash, SDS loading buffer was added to the beads to release proteins for SDS-PAGE and Western blotting.

Immunofluorescent staining
Cells were cultured on cover slips and fixed with freezing methanol after wash twice in PBS. The cover slips were then washed three times by PBS and blocked in PBS with 1% BSA for 10 min or one hour. The cover slips then were hybridized with first and second antibodies for one hour, respectively. Then the slips were mounted with prolong anti-fade kit (Invitrogen) and observed with fluorescent microscopy.

GST pulldown assays
GST-CCNE1 or GST was incubated with glutathione-sepharose beads in binding buffer (50 mM HEPES (pH 7.4), 150 mM KCl, 2.5 mM MgCl2, 5% Glycerol, 1% BSA (bovine serum albumin) at 4℃ for 1 hr. The beads were then washed twice with binding buffer, and mixed with His-tagged proteins. Samples were incubated at 4℃ for 1 h and followed by washing 3 times with binding buffer. Finally, SDS loading buffer was added and samples were heated to 95•C for 5 min for SDS-PAGE and Western blot analysis.

Reverse transcription and quantitative PCR
The indicated cells were harvested and the total RNA was extracted with RNA extraction kit (Aidlab) according to the manufacturer's manual. The amount of mRNA was quantified with Qubit (Invitrogen). Approximately 1ug of total RNA was used for reverse transcription with a first strand cDNA synthesis kit (Toyobo). Realtime PCR was then performed with My-IQ (Biorad) according to the manufacturer's standard protocol. β-actin was used to normalize the amount of each sample. Assays were repeated at least three times (n = 3). Data shown were average values ± SD.
Primer sequences are presented in Supplemental Table S1.

Protein expression by baculovirus in insect cells
Proteins was expressed and purified by using Invitrogen Bac-to-Bac® baculovirus expression system. The cDNA sequences of CUL3, RBX1 and SPOP were cloned into pFastBac vector. The plasmids were transfected into DH10BacTM cells, then plates were incubated for 48 hours at 37℃ and white colonies were picked for analysis. Then the correct bacmids were transfect into insect cells to produce recombinant baculovirus and harvested 4 days later. SF9 cells were infected by the viruses and harvested 3 days later. Protein complexes were purified with Ni Sepharose 6 Fast Flow (GE Healthcare) and Flag agarose (Sigma) sequentially.

In vitro ubiquitination assays
The

Cell proliferation assay
Cell proliferation was analyzed with MTT assay as previously described (Zhao et al., 2012). Briefly, cells were split at 1×10 3 per well in 96-well plates (n = 4).
The cells were added with MTT (0.25 μg) in each well at the indicated time points before incubation for 4 h at 37 ℃, the medium with the formazan sediment was dissolved in 50% DMF and 30% SDS (pH4.7). The absorption was read at 570 nm.

Colony formation assay
The cells were plated into a 12-well tissue culture plate (200 cells/well) and incubated at 37℃ for 8 d. The resulting colonies were rinsed with PBS, then fixed with methanol for 10 min, and stained with methylthionine chloride. The colonies in each sample were photographed and counted by FlowJo software (n = 3).

Xenograft experiments of cancer cells
The five-week-old male BALB/C nude mice were purchased from Beijing HFK bioscience Co. Ltd. A RCC mouse model was established by injecting subcutaneously the animals with 1×10 7 769-P cells in the flank region. A prostate cancer mouse model was established by injecting subcutaneously the animals with 5×10 6 DU145 cells in the flank region. Tumor volumes were measured twice a week using calipers. Tumor volumes were derived as V = 0.5×length×width 2 . After four weeks of injection, the tumors were harvested and weighed. All animal xenograft experiments were performed following the university laboratory animal guidelines and were approved by the Animal Experimentations Ethics Committee of Wuhan University.