Cancer Cell Int. 2022 Nov 15;22(1):352.
Katase N, Nishimatsu S, Yamauchi A, Okano S, Fujita S.
Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck. We identified cancer-specific genes in HNSCC and focused on DKK3 expression. DKK3 gene codes two isoforms of proteins (secreted and non-secreted) with two distinct cysteine rich domains (CRDs). It is reported that DKK3 functions as a negative regulator of oncogenic Wnt signaling and, is therefore, considered to be a tumor suppressor gene. However, our series of studies have demonstrated that DKK3 expression is specifically high in HNSCC tissues and cells, and that DKK3 might determine the malignant potentials of HNSCC cells via the activation of Akt. Further analyses strongly suggested that both secreted DKK3 and non-secreted DKK3 could activate Akt signaling in discrete ways, and consequently exert tumor promoting effects. We hypothesized that DKK3 might be a specific druggable target, and it is necessary to establish a DKK3 inhibitor that can inhibit both secreted and non-secreted isoforms of DKK3.
Methods: Using inverse polymerase chain reaction, we generated mutant expression plasmids that express DKK3 without CRD1, CRD2, or both CRD1 and CRD2 (DKK3ΔC1, DKK3ΔC2, and DKK3ΔC1ΔC2, respectively). These plasmids were then transfected into HNSCC-derived cells to determine the domain responsible for DKK3-mediated Akt activation. We designed antisense peptides using the MIMETEC program, targeting DKK3-specific amino acid sequences within CRD1 and CRD2. The structural models for peptides and DKK3 were generated using Raptor X, and then a docking simulation was performed using CluPro2. Afterward, the best set of the peptides was applied into HNSCC-derived cells, and the effects on Akt phosphorylation, cellular proliferation, invasion, and migration were assessed. We also investigated the therapeutic effects of the peptides in the xenograft models.
Results: Transfection of mutant expression plasmids and subsequent functional analyses revealed that it is necessary to delete both CRD1 and CRD2 to inhibit Akt activation and inhibition of proliferation, migration, and invasion. The inhibitory peptides for CRD1 and CRD2 of DKK3 significantly reduced the phosphorylation of Akt, and consequently suppressed cellular proliferation, migration, invasion and in vivo tumor growth at very low doses.
Conclusions: This inhibitory peptide represents a promising new therapeutic strategy for HNSCC treatment.
<Summary>
What to examine:Can we establish an antisense peptide that could suppress the protein-protein interaction of DKK3 and DKK3-driven
malignant potency of HNSCC cells ?
Results: We have established a DKK3 inhibitory peptide that can suppress tumor proliferation, migration, and invasion at low doses.
We have previously reported that DKK3 functions as tumor promoting factor which
activates Akt phosphorylation and consequently elevates cancer cell proliferation, migration,
and invasion in HNSCC cells, and that DKK3 gene has two start codons and can code both
secreted protein and non-secreted protein, and both of them independently activate Akt signaling.
Then, we conceived of antisense peptide that can inhibit protein-protein interaction of the both
isoforms of DKK3.
(1) Identification of DKK3 functional domain
We focused on the cysteine rich domain (CRD1 and CRD2) of DKK3 as a candidate
for its functional domain. Deletion mutant expression plasmids were generated and
transfected into HNSCC-derived cells, and it was shown that both of CRD1 and CRD2
are important for exertion of DKK3 function. Then, antisense peptides for the core amino
acid sequences of CRD1 and CRD2 were designed.
(2) The effects of antisense peptide for DKK3
Application of the antisense peptides in 100nM resulted in significantly reduced
Akt phosphorylation, cellular proliferation, migration, and invasion in HNSCC-derived
cells. Animal experiments revealed that the peptides could suppress the in vivo
tumor growth of the HNSCC-derived cells in the xenograft model.
In conclusion, we have established a DKK3 specific inhibitory peptide that can suppress tumor proliferation, migration, and invasion at low doses. We will continue to investigate the application of this peptide in a clinical setting.