USP25 maintains KRAS expression and inhibiting the deubiquitinase suppresses KRAS signaling in human cancer

Abstract

KRAS is a prominent oncogene mutated in a large number of human malignancies, particularly in pancreatic, colorectal, and lung tumors. We demonstrate here that KRAS, including its various activating mutants, is subjected to ubiquitin-mediated proteasomal degradation in cancer cells. Through an siRNA-based screening of deubiquitinases, we identified USP25 as a deubiquitinase for KRAS. Depleting USP25 expression increases ubiquitination and proteasomal degradation of KRAS, leading to the suppression of its oncogenic activity. We further show that USP25 inhibitors we have discovered are capable of destabilizing KRAS in cancer cells and are efficacious in blocking tumor xenograft growth in mice. These findings provide evidence supporting the notion that targeting the deubiquitinase USP25 can effectively, albeit indirectly, suppress KRAS and potentially aid in the treatment of tumors driven by KRAS-activating mutations.

Keywords: KRAS; USP25; proteasomal degradation; ubiquitination.

Figure 1

USP25 is a deubiquitinase for KRAS.A, USP25 deubiquitinates KRAS. KRAS ubiquitination levels were assessed via immunoprecipitation and Western blotting following the knockdown of USP25 with two independent shRNAs in HCT116 cells. The amounts of total proteins from USP25-depleted cells were adjusted for immunoprecipitation to reflect the fact that these cells now contained less KRAS protein. B, coimmunoprecipitation analysis of the interaction between HA-tagged KRAS and the endogenous USP25 in HEK293T cells. C, coimmunoprecipitation analysis of the interaction between HA-tagged USP25 and the endogenous KRAS in HEK293T cells. D, GST pull-down assay with GST-KRAS (full length and truncations) and His-USP25. A diagram of KRAS protein is presented. His-USP25 was detected with Western blotting (upper panel). The GST and GST fusion proteins were separated in an SDS-PAGE gel and visualized with Coomassie blue staining (lower panel). E, coimmunoprecipitation analysis of the interaction between HA-tagged KRAS (WT and the 8A mutant, D) and the endogenous USP25 in HEK293T cells. F, analysis of the ubiquitination levels in KRAS (WT and the 8A mutant, D) in 293T cells with or without USP25 overexpression. GST, glutathione-S-transferase.

Figure 1

USP25 is a deubiquitinase for KRAS.A, USP25 deubiquitinates KRAS. KRAS ubiquitination levels were assessed via immunoprecipitation and Western blotting following the knockdown of USP25 with two independent shRNAs in HCT116 cells. The amounts of total proteins from USP25-depleted cells were adjusted for immunoprecipitation to reflect the fact that these cells now contained less KRAS protein. B, coimmunoprecipitation analysis of the interaction between HA-tagged KRAS and the endogenous USP25 in HEK293T cells. C, coimmunoprecipitation analysis of the interaction between HA-tagged USP25 and the endogenous KRAS in HEK293T cells. D, GST pull-down assay with GST-KRAS (full length and truncations) and His-USP25. A diagram of KRAS protein is presented. His-USP25 was detected with Western blotting (upper panel). The GST and GST fusion proteins were separated in an SDS-PAGE gel and visualized with Coomassie blue staining (lower panel). E, coimmunoprecipitation analysis of the interaction between HA-tagged KRAS (WT and the 8A mutant, D) and the endogenous USP25 in HEK293T cells. F, analysis of the ubiquitination levels in KRAS (WT and the 8A mutant, D) in 293T cells with or without USP25 overexpression. GST, glutathione-S-transferase.

Figure 2

Identification of the ubiquitination sites in KRAS.A, the hypervariable regions of KRAS4A and KRAS4B. The red highlighted lysine residues are responsible for KRAS ubiquitination. B, analysis of the ubiquitination levels in KRAS4B mutants. Flag-tagged WT KRAS4B, KRAS4B^K169R, or KRAS4B^K172R were expressed in HEK293T cells and immunoprecipitated for Western blotting analysis of ubiquitination levels. C, KRAS4B^K172R no longer requires USP25 for stable expression. HEK293T cells were first transfected with Flag-tagged KRAS4B WT or K172R mutant and divided into two parts which were then infected with lentiviruses carrying a shUSP25-expressing cassette or shNC control cassette. Flag-tagged KRAS4B protein levels were analyzed with Western blotting. D, analysis of ubiquitination levels in KRAS4A mutants. Flag-tagged WT KRAS4A, KRAS4A^K169R, or KRAS4A^K172R were expressed in HEK293T cells and immunoprecipitated for Western blotting analysis of ubiquitination levels. E, KRAS4A^K169R no longer requires USP25 for stable expression. HEK293T cells were first transfected with Flag-tagged KRAS4A WT or K169R mutant and divided into two parts which were then infected with lentiviruses carrying a shUSP25-expressing cassette or shNC control cassette. Flag-tagged KRAS4A protein levels were analyzed with Western blotting.

Figure 3

The proliferation of tumor cells requires USP25.A, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown in HCT116 cells. B, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-1 and shUSP25-2 and re-expression of USP25 in HCT116 cells. C, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-2 and exogenous expression of KRAS^G13D in HCT116 cells. D, growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-1) or depleted plus re-expression of USP25. E, growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-2) or depleted plus exogenous expression of KRAS^G13D. F, the growth of the xenograft tumors derived from HCT116 cells carrying doxycycline-inducible shUSP25 expression cassette (Tet-on-shUSP25) or a negative control (Tet-on-shNC). G, photographic representation of tumors excised from the nude mice. H, the weight of tumors in (C). I, Western blotting analysis for KRAS and its downstream signaling proteins in the tumor samples. J, immunohistochemical (IHC) analysis of the expression of proliferation marker Ki67 and phosphor-ERK (p-ERK) in tumor tissue sections along with quantitation of the IHC staining. One section from each one of the 5 tumor samples was stained and quantitated. Data are presented as mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Figure 3

The proliferation of tumor cells requires USP25.A, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown in HCT116 cells. B, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-1 and shUSP25-2 and re-expression of USP25 in HCT116 cells. C, Western blotting analysis of the expression levels of KRAS and its downstream signaling pathway proteins following USP25 knockdown with shUSP25-2 and exogenous expression of KRAS^G13D in HCT116 cells. D, growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-1) or depleted plus re-expression of USP25. E, growth curve analysis of HCT116 cells with USP25 expression depleted (with shUSP25-2) or depleted plus exogenous expression of KRAS^G13D. F, the growth of the xenograft tumors derived from HCT116 cells carrying doxycycline-inducible shUSP25 expression cassette (Tet-on-shUSP25) or a negative control (Tet-on-shNC). G, photographic representation of tumors excised from the nude mice. H, the weight of tumors in (C). I, Western blotting analysis for KRAS and its downstream signaling proteins in the tumor samples. J, immunohistochemical (IHC) analysis of the expression of proliferation marker Ki67 and phosphor-ERK (p-ERK) in tumor tissue sections along with quantitation of the IHC staining. One section from each one of the 5 tumor samples was stained and quantitated. Data are presented as mean ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Figure 4

Pharmacological inhibition of USP25 downregulates KRAS expression and its downstream signaling.A, analysis of KRAS signaling pathway proteins with Western blotting analysis in HT29 cells treated with different concentrations of CT1113 for 72 h. B, Western blotting analysis of KRAS signaling pathway proteins across multiple cell lines treated with 800 nM CT1113 for 72 h. C, examination of KRAS signaling pathway proteins with Western blotting analysis in H23 cells treated with CT1113 and ARS-1620 individually or in combination for 72 h. Since CT1113 also inhibits USP28, the expression levels of USP28 and its substrate c-MYC were also examined. D, examination of KRAS signaling pathway proteins with Western blotting analysis in PANC1 cells treated with CT1113 and MRTX1133 individually or in combination for 72 h. The expression levels of USP28 and its substrate c-MYC were also examined.

Figure 5

Pharmacological inhibition of USP25 suppresses tumor growth.A, photographs of the xenograft tumors derived from human pancreatic cancer cell line SW1990. 1 × 10⁶ SW1990 cells were inoculated in each nude mouse. When the xenografts grew to palpable sizes, the mice were given CT1113 orally (20 or 25 mg/kg body weight) twice a day for 2 weeks. B, the weight of the tumors in (A). C, Western blotting analysis of KRAS signaling pathway proteins in vehicle control and CT1113 (25 mg/kg) treated tumors. Data are presented as mean ± SD. ∗∗∗∗p < 0.0001.

Figure 6

The expression of USP25 correlates with RAS in human cancer.A, representative immunohistochemical staining images from tissue microarrays (TMAs) for USP25 and RAS proteins. The human lung and colon adenocarcinoma TMAs consist of 80 pairs of cancerous and para-cancerous tissues. The human pancreatic cancer TMA consists of cancerous tissues only. The scale bar represents 100 μm. B, the correlation between USP25 and RAS expression in human cancers. The expression scores of the two proteins in TMAs were plotted against each other. C, enhanced expression of USP25 and RAS in human cancer specimens relative to adjacent nontumor tissues. Shown are representative images of IHC staining of USP25 and RAS in cancerous and para-cancerous tissues of the lung cancer and colon adenocarcinoma samples. No para-cancerous tissues are available in the pancreatic cancer samples for comparison. D, pair-wise comparison of USP25 and RAS expression in cancerous and para-cancerous tissues from (C). IHC, immunohistochemical.

Figure 7

A schematic illustration of the regulation of KRAS by USP25. The expression of KRAS proteins depends on USP25. Compromising USP25 function can lead to suppression of KRAS.