A cell-permeable peptide-based PROTAC against the oncoprotein CREPT proficiently inhibits pancreatic cancer

Abstract

Cancers remain a threat to human health due to the lack of effective therapeutic strategies. Great effort has been devoted to the discovery of drug targets to treat cancers, but novel oncoproteins still need to be unveiled for efficient therapy. Methods: We show that CREPT is highly expressed in pancreatic cancer and is associated with poor disease-free survival. CREPT overexpression promotes but CREPT deletion blocks colony formation and proliferation of pancreatic cancer cells. To provide a proof of concept for CREPT as a new target for the inhibition of pancreatic cancer, we designed a cell-permeable peptide-based proteolysis targeting chimera (PROTAC), named PRTC, based on the homodimerized leucine-zipper-like motif in the C-terminus domain of CREPT to induce its degradation in vivo. Results: PRTC has high affinity for CREPT, with Kd = 0.34 +/- 0.11 μM and is able to permeate into cells because of the attached membrane-transportable peptide RRRRK. PRTC effectively induces CREPT degradation in a proteasome-dependent manner. Intriguingly, PRTC inhibits colony formation, cell proliferation, and motility in pancreatic cancer cells and ultimately impairs xenograft tumor growth, comparable to the effect of CREPT deletion. Conclusions: PRTC-induced degradation of CREPT leads to inhibition of tumor growth, which is promising for the development of new drugs against pancreatic cancer. In addition, using an interacting motif based on the dimerized structure of proteins may be a new way to design a PROTAC aiming at degrading any protein without known interacting small molecules or peptides.

Keywords: CREPT; PROTAC; degradation; drug target; pancreatic cancer.

Figure 1

Overexpression of CREPT in pancreatic cancer promotes cell proliferation and tumorigenesis. (A) Ectopic expression of CREPT in K-ras/p53-driven pancreatic cancer tissues stained with anti-CREPT antibody. Scale bars, 100 μm. (B) Ectopic expression of CREPT in human pancreatic tumor tissues stained with anti-CREPT antibody. Scale bars, 50 μm. (C) Kaplan-Meier plot of cumulative disease-free survival (DFS) of 182 pancreatic adenocarcinoma samples in TCGA database. (CREPT high-expression group, purple line; CREPT low-expression group, blue line). The p-value obtained by comparing the two survival curves was 0.0154. (D-F) Overexpression of CREPT significantly increased the number of colonies and promoted cell viability. (G-I) Deletion of CREPT dramatically reduced the number of colonies and inhibited cell viability. The results are represented as the mean ± SD from three independent repeats.

Figure 2

Design and evaluation of PRTC. (A) Graphic illustration of the CREPT structure. RPR, regulation of nuclear pre-mRNA. CCT, coiled-coil terminus. (B) GST pull-down experiments on Flag-CREPT with purified GST-tagged proteins from HEK293T cells (left) and E. coli (right). (C) Exogenous IP experiment with Flag-CREPT and Myc-CREPT in HEK293T cells. (D) The CCT domain is essential for the formation of homodimers. (E) Exogenous IP experiment of Flag-CREPT with HA-CREPT or HA-CREPT-m, which is a mutant in which residues leucine 269, 276, and 283 are replaced with proline residues. (F) CD spectroscopy assay of PRTC, PRTC-m and PRTC-v. The positions of 192 nm, 208 nm and 222 nm wavelengths are marked as black dash line. All peptides were dissolved in deionized water at a final concentration of 0.1 mg/mL. (G) Schematic diagram of PRTC. CREPT ligand (CL), a polypeptide from amino acids lysine 266 to valine 286. IYP (OH) AL, named VHL ligand. 6-Aminohexanoic acid (AHX) is the linker to bridge the CREPT ligand and VHL ligand. P (OH) is trans-4-hydroxy-L-proline. The pentapeptide (RRRRK) on the C-terminus of PRTC assists PRTC translocation into cells.

Figure 3

Identification of the permeability of PRTC in Panc-1 cells. (A) Entry of FITC- labeled peptides into Panc-1 cells tested by flow cytometry. FITC-TAT is the positive control. Measurements were performed in triplicate. (B) Quantification of cellular uptake of Panc-1 cells treated with different concentrations of FITC-PRTC. (C) Confocal microscope images of Panc-1 cells treated with 10 μM FITC labeled peptides. Scale bars, 10 μm. (D) Confocal microscope images of Panc-1 cells treated with different dosages of FITC-PRTC. Scale bars, 10 μm.

Figure 4

PRTC is able to interact with CREPT. (A) Molecular stimulation of the PRTC binding CREPT CCT domain using Schrodinger (Maestro 11.8) and SWISS-MODEL. (B) Microscale thermophoresis result of FITC-labeled PRTC with GST-tagged CREPT. Kd = 0.34 ± 0.11 μM. (PRTC, blue dot; PRTC-m, green dot). (C) Thermal shift assay results of GST-tagged CREPT with deionized H₂O (Ctrl), PRTC or PRTC-m. PRTC induced 6 °C shift in T_m compared to the control group. (D) Exogenous IP experiment of GST-tagged CREPT Ligand (CL) with Myc-CREPT and Myc-CREPT-CCT. (E) Exogenous IP experiment of Flag-CREPT with GST-CL or GST-CL-m in HEK293T cells. (F) GST pull-down experiments of Flag-CREPT with GST-tagged CL or CL-m using purified proteins from HEK293T cells.

Figure 5

PRTC induces the ubiquitination and proteasome- dependent degradation of the endogenous CREPT protein. (A) Immunoblot of CREPT following 24 h incubation with different dosages of PRTC or PRTC-m in Panc-1 cells. (B) Immunoblot of CREPT exposed to 10 μM PRTC or PRTC-m for different times in Panc-1 cells. (C) Western blot analysis of CREPT following 24 h incubation with 10 μM PTRC in Panc-1 cells. MG132 is an inhibitor of proteasome activity. (D) Immunofluorescence visualization of Panc-1 cells after treatment with PRTC for 24 h. Cells were stained with anti-CREPT antibody. Scale bars, 20 μm.

Figure 6

The degradation of CREPT by PRTC is dependent on its VHL ligand. (A) Immunoblot of CREPT following 24 h incubation with different dosages of PRTC or PRTC-v in Panc-1 cells. (B) Immunoblot of CREPT exposed to 10 μM PRTC or PRTC-v for different time points in Panc-1 cells. (C) Endogenous IP experiment of CREPT with ubiquitin treated with 10 μM PTRC, PTRC-m or PTRC-v in Panc-1 cells. IP: CREPT, IB: Anti-ubiquitin.

Figure 7

CREPT degradation by PRTC treatment inhibits cell proliferation and tumorigenesis in pancreatic cancer. (A) CCK-8 assay of wild-type Panc-1 cells treated with deionized H₂O (Ctrl), CREPT deletion cells treated with deionized H₂O (CREPT-/-), Panc-1 cells treated with 10 μM PRTC (PRTC) and Panc-1 cells treated with 10 μM PRTC-m (PRTC-m). (B-C) Colony formation of Ctrl, CREPT-/-, PRTC and PRTC-m. (D-E) Wound healing assay of Ctrl, CREPT-/-, PRTC and PRTC-m. (F) Xenograft tumor formation of Ctrl, CREPT-/-, PRTC group. 5×10⁶ wild-type Panc-1 cells or CREPT knockout Panc-1 cells were subcutaneously injected into 4-week-old female Balb/c nude mice. Mice bearing tumors were randomly divided into two groups and intraperitoneally administered control solvent (0.9% saline) or PRTC (10 mg/kg) every 2 days for 4 weeks. (G) Tumor weights of xenograft tumors. (H) Body weight curves of Ctrl, CREPT-/-, PRTC group. n=5. The values were measured every week after treated with 0.9% saline or PRTC.

Figure 8

Schematic diagram of PRTC degrading the oncoprotein CREPT. PRTC permeates into pancreatic cancer cells and competitively binds dimerized CREPT. Meanwhile, PRTC binds an E3-ubiquitin ligase complex. The formation of the trimeric complex contributes to the transfer of ubiquitins to the CREPT. The poly-ubiquitinated CREPT is recognized by the proteasome and degraded. PRTC proficiently inhibits colony formation, cell proliferation, and motility in pancreatic cancer cells and ultimately impairs xenograft tumor growth. TA, the targeting arm of PRTC. DA, the degrading arm of PRTC. Ub, ubiquitin.