Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 12;7(28):44299-44309.
doi: 10.18632/oncotarget.9996.

A recombinant chimeric protein specifically induces mutant KRAS degradation and potently inhibits pancreatic tumor growth

Ting Pan  1   2   3 , Yiwen Zhang  1   2   3 , Nan Zhou  1   2   3 , Xin He  1   2   3 , Cancan Chen  1   2   3 , Liting Liang  1   3 , Xiaobing Duan  1   2 , Yingtong Lin  1   3 , Kang Wu  1   2   3 , Hui Zhang  1   2   3
Affiliations

A recombinant chimeric protein specifically induces mutant KRAS degradation and potently inhibits pancreatic tumor growth

Ting Pan et al. Oncotarget. .

Abstract

Pancreatic cancer is one of the most lethal human diseases, with an all-stage 5-year survival rate below 5%. To date, no effective and specific therapy is available for this disease. Mutations in KRAS are frequently reported in pancreatic and many other cancers; thus, KRAS is an attractive therapeutic target. Our objective was to specifically eliminate mutant KRAS and induce cell death of tumors expressing this mutant protein. We thus constructed several chimeric proteins by connecting the C-terminal domains of several adaptor proteins of E3 ubiquitin ligases such as CBL, CHIP, E6AP, and VHL, as well as VIF encoded by human immunodeficiency virus type 1 (HIV-1), to the Ras binding domain (RBD) of Raf. Although all of these chimeric proteins caused the degradation of mutant KRAS and the death of KRAS-mutant-tumor cell lines, the RBD-VIF with a protein transduction domain (PTD), named PTD-RBD-VIF, had the strongest tumor-killing effect. Intraperitoneally administered recombinant PTD-RBD-VIF potently inhibited the growth of xenografted KRAS-mutant pancreatic cancer cells. Our findings indicate that recombinant PTD-RBD-VIF, a chimeric protein with a combined cellular-viral origin, could be further developed for the treatment of various tumors harboring mutant or over-activated KRAS, especially for cases presenting with pancreatic cancer recurrence after surgery.

Keywords: KRAS; Ras binding domain; Vif; pancreatic cancer; ubiquitin.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no conflicts of interest exists.

Figures

Figure 1
Figure 1. Different chimeric proteins significantly inhibit the expression of mutant KRASG12D or KRASG12V
(A) Schematic of construction of different chimeric proteins and mutant KRAS-RFP-harboring plasmids. (B) Different plasmids harboring various chimeric proteins were co-transfected with KRASG12D or KRASG12V RFP-expressing plasmids into HEK293T cells. After 48 h, KRAS-RFP expression was detected under fluorescence microscope. (C) The MFI results were evaluated by flow cytometry and the mean ± SEM are shown. Error bars indicate SEM. *p < 0.05, **p < 0.01 compared to controls. (D) KRASG12D or KRASD12V RFP-expressing plasmids were co-transfected with high or low doses of adaptor plasmids into HEK293T cells, and the levels of mutant KRAS were determined by western blotting after 48 h.
Figure 2
Figure 2. Purified chimeric proteins induce cell death in tumor cell lines
(A) Schematic of construction of different chimeric proteins. (B) The purified proteins were added to different cell cultures. After 48 h, cells were examined under microscope. (C) The percentage of cell death was evaluated by MTT assay, and mean ± SEM is shown. Error bars indicate SEM. (D) Different doses of chimeric proteins were added to Panc-1 cells and IC50 was calculated. Error bars indicate SEM.
Figure 3
Figure 3. Purified PTD-RBD-VIF has low immunogenicity and toxicity
(A) Different chimeric proteins (PTD-RBD-CHIP, PTD-RBD-E6AP, PTD-RBD-VIF) were intraperitoneally injected into 6 mice individually and the immunogenicity of each protein was detected by ELISA after 4 wk. Error bars indicate SEM. (B–D) The safety of PTD-RBD-VIF was examined by the acute toxicity test. (B) Vital organs, including spleen, lung, liver, kidney, and heart, from mice treated with various doses of PTD-RBD-VIF were histologically sectioned and stained with HE. (C) Weight of male BALB/c mice after intraperitoneal injection of PTD-RBD-VIF. Error bars indicate SEM. (D) The effect of PTD-RBD-VIF on the hepatic and renal functions of mice. Error bars indicate SEM. (BUN: Blood urea nitrogen, CRE: Creatine, AST: Aspartate transaminase, ALT: Alanine transaminase).
Figure 4
Figure 4. RBD-VIF mediates the degradation of mutant KRAS through Vif-mediated ubiquitin system and inhibits the downstream of MAPK-ERK pathway
(A) RBD-VIF-HA plasmid and KRAS plasmid were co-transfected into HEK293T cells, and 48 h later, cells were collected for co-immunoprecipitation experiments. GFP-HA plasmid was transfected as a negative control. (B) RBD-VIF-HA plasmid and mutant KRAS plasmid were co-transfected into HEK293T cells and the culture was maintained for 12 h with or without 2 μM MG-132 before harvesting the cells for western blot analysis at 48 h. (C) Mutant KRAS-RFP-expressing plasmids were co-transfected with si-NC, si-Elongin B, si-Elongin C, or si-Cullin 5 respectively into HEK293T cells. After 48 h, the expression of KRAS-RFP was detected under fluorescence microscope. (D) Panc-1 cells were treated with RBD-VIF or a control protein (PTD-GFP). After 24 h, cells were harvested for western blot analysis to detect the phosphorylation of ERK1/2. (E) Panc-1 cells were treated with RBD-VIF or the control protein PTD-GFP for 24 h and then the cells were harvested for western blot to detect P-MEK expression.
Figure 5
Figure 5. Comparison of PTD-RBD-VIF with different ways of administrations in nude mice
(A–C) Xenografts were established using Panc-1 cell line in female BALB/c nude mice. Four groups were randomly assigned and injected with control protein or recombinant PTD-RBD-VIF by tail vein injection, intraperitoneal injection and orthotopic injection every 3 days (n = 3). (A) Tumor volumes were measured every 5 days. Error bars indicate SEM. (B) Tumor formation assay to evaluate the effect of different PTD-RBD-VIF delivery pathways. (C) The data represent mean ± SEM (n = 3). Error bars indicate SEM. (D–E) Xenografts were established using BxPC-3 cell line in female BALB/c nude mice. Two groups were randomly assigned and injected with control protein or recombinant PTD-RBD-VIF by tail vein injection every 3 days (n = 3). (D) Tumor formation assay to evaluate the effect of different PTD-RBD-VIF delivery pathways. (E) The data represent mean ± SEM (n = 3). Error bars indicate SEM.
Figure 6
Figure 6. PTD-RBD-VIF potently inhibits pancreatic tumor growth with intraperitoneal injection in vivo
Xenografts were established using Panc-1 cell line in female BALB/c nude mice. Two groups were randomly assigned and injected (i.p.) with recombinant PTD-RBD-VIF or control protein every 3 days (n = 6). (A) Tumor volumes were measured every 5 days. Error bars indicate SEM. (B) Tumor formation assay to evaluate the effect of PTD-RBD-VIF or control protein. (C) The data represent mean ± SEM (n = 6). Error bars indicate SEM. (D) Survival curve for 2 groups of mice. (E) Representative western blot to detect the expression of KRAS in the tumors in these 2 groups. (F) Representative TUNEL staining of tumors in each group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y, Adenis A, Raoul JL, Gourgou-Bourgade S, de la Fouchardiere C, Bennouna J, Bachet JB, Khemissa-Akouz F, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. The New England journal of medicine. 2011;364:1817–1825. - PubMed
    1. Campbell PJ, Yachida S, Mudie LJ, Stephens PJ, Pleasance ED, Stebbings LA, Morsberger LA, Latimer C, McLaren S, Lin ML, McBride DJ, Varela I, Nik-Zainal SA, et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature. 2010;467:1109–1113. - PMC - PubMed
    1. Hidalgo M. Pancreatic cancer. The New England journal of medicine. 2010;362:1605–1617. - PubMed
    1. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Kamiyama H, Jimeno A, Hong SM, Fu B, Lin MT, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008;321:1801–1806. - PMC - PubMed
    1. Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, Miller DK, Wilson PJ, Patch AM, Wu J, Chang DK, Cowley MJ, Gardiner BB, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491:399–405. - PMC - PubMed

MeSH terms

Substances