HOOK1 Inhibits the Progression of Renal Cell Carcinoma via TGF‐β and TNFSF13B/VEGF‐A Axis

Abstract Accumulating evidence shows HOOK1 disordered in human malignancies. However, the clinicopathological and biological significance of HOOK1 in renal cell carcinoma (RCC) remains rarely studied. In this study, the authors demonstrate that HOOK1 is downregulated in RCC samples with predicted poorer clinical prognosis. Mechanistically, HOOK1 inhibits tumor growth and metastasis via canonical TGF‐β/ALK5/p‐Smad3 and non‐canonical TGF‐β/MEK/ERK/c‐Myc pathway. At the same time, HOOK1 inhibits RCC angiogenesis and sunitinib resistance by promoting degradation of TNFSF13B through the ubiquitin‐proteasome pathway. In addition, HOOK1 is transcriptionally regulated by nuclear factor E2F3 in VHL dependent manner. Notably, an agonist of HOOK1, meletin, is screened and it shows antitumor activity more effectively when combined with sunitinib or nivolumab than it is used alone. The findings reveal a pivotal role of HOOK1 in anti‐cancer treatment, and identify a novel therapeutic strategy for renal cell carcinoma.


RNA extraction and quantitative real-time PCR analysis.
Total RNA was extracted using an RNeasy mini kit (Qiagen, Germany). The RNA concentration was measured using Nanodrop 2000 (Thermo Scientific, USA). cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen) with the primers (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. β-actin was used to correct the difference in template input. The relative RNA expression was calculated using the 2-ΔCT method. The following primers were used in the present study: HOOK1, 5′-CAGACATTCAATACTGCCTCACC-3′

Western blot analysis.
Briefly speaking, cells were lysed in RIPA lysis buffer containing PMSF on ice. The concentrations of proteins were determined using a BCA protein kit (Beyotime, China) and whole lysates were mixed with 6 X SDS loading buffer, heated at 100 °C for 10 min. A total of 30 mg of protein from each sample were loaded, running and then transferred to PVDF membranes. Immunoreactive bands were visualized using ECL western blot kit.

Migration and invasion assays.
Transwell assays were performed to evaluate migration and invasion activities.

Cell-adhesion assays.
Cell-adhesion assays were performed using the CytoSelect 48-Well Cell Adhesion Assay kit (fibronectin coated) according to manufacturer's instructions (Cell Biolabs, San Diego, CA).

Tube formation assay.
96-well plates and tips were incubated at -20℃ for 30 m and Matrigel (BD) was transferred onto ice. Matrigel (10 mL) was added to each well with the cool tip and distributed evenly by shaking the plate. The plate was placed on ice for 2 min and then incubated in a cell culture incubator for 30 min. Cell suspension was made by trypsinization and adjusted to 1x105 cells/mL after live cell counting. Cells were added into the pre-incubated plate at 100 mL/well, and incubated under normal conditions. Images were acquired on microscopic 6 h later. Then the enclosed networks of complete tubes from randomly chosen fields were counted and averaged.

Plasmids, virus production and transfection.
To overexpress HOOK1, E2F3, HIF-1α and HIF-2α, cDNA of those genes was cloned into pWPI vector and the silence sequence was cloned into PLKO.

Luciferase assay
Interactions between E2FF3 and HOOK1 in RCC were verified by luciferase assay according to previous studies. Briefly, Human HOOK1 promoter region was constructed into pGL3-basic vector (Promega, USA). Then cells were seeded in 24-well plates and transfected with cDNA via Lipofectamine 3000 (Invitrogen).
pRL-TK was used as negative control. The luciferase activity measured were based on the manufacturer's manual (Promega).
The intensity of staining was determined as: 0 (no staining); 1 (weak staining = light yellow); 2 (moderate staining = yellow brown); and 3 (strong staining = brown). A semiquantitative scoring criterion was used, in which the staining index (values 0-12) were calculated by multiplying the staining intensity and the positive cells proportion.
Finally, cases were classified into two different groups: low expression cases (score 0-6) and cases with high expression (score 7-12).

Multiplex immunofluorescence staining and analysis
mIHC was performed as previous described [1]. Formalin-fixed paraffin-embedded

Protein-protein docking and molecular docking
The protein crystal structure of HOOK1 and TNFSF13B was downloaded from the