Salt‐Inducible Kinase 2‐Triggered Release of Its Inhibitor from Hydrogel to Suppress Ovarian Cancer Metastasis

Abstract Salt‐inducible kinase 2 (SIK2) is a promising target for ovarian cancer therapy due to its critical role in tumorigenesis and progression. Currently available SIK2 inhibitors have shown remarkable therapeutic effects on ovarian cancers in preclinical studies. However, direct administration of the SIK2 inhibitors may bring significant off‐target effect, limiting their clinical applications. In this work, by rational design of a hydrogelator Nap‐Phe‐Phe‐Glu‐Glu‐Leu‐Tyr‐Arg‐Thr‐Gln‐Ser‐Ser‐Ser‐Asn‐Leu‐OH (Nap‐S) to coassemble a SIK2 inhibitor HG‐9‐91‐01 (HG), a SIK2‐responsive supramolecular hydrogel (Gel Nap‐S+HG) for local administration and SIK2‐responsive release of HG is reported to efficiently suppress ovarian cancer metastasis. Under the activation of SIK2 overexpressed in ovarian cancers, Nap‐S in the hydrogel is phosphorylated to yield hydrophilic Nap‐Phe‐Phe‐Glu‐Glu‐Leu‐Tyr‐Arg‐Thr‐Gln‐Ser(H2PO3)‐Ser‐Ser‐Asn‐Leu (Nap‐Sp), triggering the disassembly of the hydrogel and a responsive release of the inhibitor. Cell experiments indicate that sustained release of HG from Gel Nap‐S+HG induce a prominent therapeutic effect on cancer cells by inhibiting SIK2 and phosphorylation of their downstream signaling molecules. Animal experiments demonstrate that, compared with those tumor model mice treated with free HG, Gel Nap‐S+HG‐treatment mice show an enhanced inhibition on ovarian tumor growth and metastasis. It is anticipated that the Gel Nap‐S+HG can be applied for ovarian cancer therapy in clinic in the near future.


Quantitative Real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and reverse transcribed using StarScript II First-strand cDNA Synthesis Kit-II (GenStar, A214-02, China) following the manufacturer's protocols. Quantitative real-time was performed using a SYBR Green PCR kit (GeneStar, A303-10, China) in a 20 µL qPCR reaction according to the manufacturer's protocols. Each sample was tested in triplicate. β-actin was used as an internal control for all samples. Primer sequences are listed in the table below.
Primer set name Forward Sequence Reverse Sequence SIK2 cagcagctgcaggaacatag gacttggctgtgggtaggag β-actin tcgcctttgccgatccg atgatctgggtcatcttcycg SIK2-Instrusted Disassembly of Hydrogels in Vitro 100 µL SKOv3-SIK2 cell lysates (4 × 10 5 cells) were added an equal volume of Gel Nap-S and Gel Nap-S+HG at 37°C. After incubation overnight, Gel Nap-S and Gel Nap-S+HG were partially disassembled by SIK2 in the cell lysates.

CCK-8 Assay
SKOv3-SIK2 cells were seeded in 96-well plate (2,000 cells/well). Culture media (Ctrl group), Gel Nap-S (530 µM Nap-S), HG (4.24 µM) or Gel Nap-S+HG (530 µM Nap-S + 4.24 µM HG) was added into the plates, respectively. At day 1, 2 and 3, cell proliferation was detected using the CCK-8 assay according to the manufacturer's protocols. Briefly, the culture medium in each well was replaced with 100 µL DMEM containing 10 µL CCK-8. After incubation for 1 h at 37 °C, optical density (OD) values of absorbance at 450 nm were measured in a microplate reader (SpectraMax iD3, Molecular Devices). Each of the experiments was performed at least three times.

Cell Apoptosis Assays
As SKOv3-SIK2 cells were carried with GFP-tag, which could interfere with the green fluorescent signal, Annexin V-PE Apoptosis Detection Kit (Beyotime, C1065M, China) was added to testify the apoptotic cells at different treatments (DMEM, Nap-S, HG and Nap-S+HG). After incubation for 24h, apoptotic SKOv3-SIK2 cells were stained red and observed with fluorescence microscope (Nicon, Japan).

In Vitro Migration and Invasive Assays
To detect the migration and invasive capacity of SKOv3-SIK2 cells after different treatment, the scratch wound healing and transwell invasive assays were performed. For wound healing assays, SKOv3-SIK2 cells were seeded in a 6-well plate (2×10 4 cells/well) and then incubated with DMEM, Gel Nap-S (530 µM Nap-S), HG (4.24 µM) and Gel Nap-S+HG (530 µM Nap-S + 4.24 µM HG) for 24 h. A scratch with 200 µL yellow pipette tip was placed in the middle of the wells. Then the wells were gently washed with PBS to remove the cell debris. Afterwards, each well was added with incomplete culture medium and photographed at 0 h and 24 h. For transwell invasive assays, after treatment with DMEM, Gel Nap-S, HG and Gel Nap-S+HG for 24 h, SKOv3-SIK2 cells in each group were digested with trypsin and then seeded to the upper chamber at the density of 2 × 10 4 /100 µL (dispersed in FBS-free DMEM). The lower chamber was added with 550 µL DMEM containing 20% FBS. After 24 h incubation, the chambers were washed with PBS from upper to lower twice gently, and then the migrated cells in the lower chambers were fixed with 70% (v/v) ethanol solutions for 30 min. Then the chambers were stained with 0.1% (w/v) crystal violet solution for 30 min. Later, the cells in the upper chamber were gently wiped off with a cotton swab soaked in PBS. Migrated cells were observed with microscope (XD-202, Nanjing Jiangnan Yongxin Optical Co, Ltd.). Next, the crystal violet was eluted by a 33% acetic acid solution and measured at the absorbance of 570 nm.

Western Blot Analyses
SKOv3-SIK2 cells were cultured with DMEM, Gel Nap-S (Nap-S: 530 µM), HG (4.24 µM), Gel Nap-S+HG (530 µM Nap-S + 4.24 µM HG) and complete medium only for 24 hours. Afterwards, total proteins were extracted from the cells and separated by electrophoresis using 6.5%-10% SDS-PAGE gels, then transferred onto PVDF membranes (Millipore, USA). The membranes were incubated with specific primary antibodies overnight at 4 °C and HRPconjugated second antibody for 1 hour at room temperature. GAPDH was used as a loading control. Proteins were visualized using an enhanced chemiluminescence system (Millipore, USA

Syntheses of Nap-S and Nap-Sp
Scheme S1. The synthetic route for Nap-S.