GRP78 inhibitor YUM70 upregulates 4E-BP1 and suppresses c-MYC expression and viability of oncogenic c-MYC tumors

Abstract

The 78-kDa glucose regulated protein (GRP78) commonly upregulated in a wide variety of tumors is an important prognostic marker and a promising target for suppressing tumorigenesis and treatment resistance. While GRP78 is well established as a major endoplasmic reticulum (ER) chaperone with anti-apoptotic properties and a master regulator of the unfolded protein response, its new role as a regulator of oncoprotein expression is just emerging. MYC is dysregulated in about 70 % of human cancers and is the most commonly activated oncoprotein. However, despite recent advances, therapeutic targeting of MYC remains challenging. Here we identify GRP78 as a new target for suppression of MYC expression. Using multiple MYC-dependent cancer models including head and neck squamous cell carcinoma and their cisplatin-resistant clones, breast and pancreatic adenocarcinoma, our studies revealed that GRP78 knockdown by siRNA or inhibition of its activity by small molecule inhibitors (YUM70 or HA15) reduced c-MYC expression, leading to onset of apoptosis and loss of cell viability. This was observed in 2D cell culture, 3D spheroid and in xenograft models. Mechanistically, we determined that the suppression of c-MYC is at the post-transcriptional level and that YUM70 and HA15 treatment potently upregulated the eukaryotic translation inhibitor 4E-BP1, which targets eIF4E critical for c-MYC translation initiation. Furthermore, knock-down of 4E-BP1 via siRNA rescued YUM70-mediated c-MYC suppression. As YUM70 is also capable of suppressing N-MYC expression, this study offers a new approach to suppress MYC protein expression through knockdown or inhibition of GRP78.

Keywords: 4E-BP1; Breast cancer; GRP78; Head and neck cancer; MYC; Pancreatic cancer; Small molecule inhibitors; YUM70.

Fig. 1

GRP78 deficiency reduces c-MYC expression in human HNSCC and their cisplatin-resistant derivatives. The HNSCC cells SCC25 (A), SCC15 (B), the cisplatin-resistant clonal lines cisR-2 (C), and cisR-3 (D) were treated with control siRNA (siCtrl) or siRNA targeting GRP78 (si78) for 48 h. Whole cell lysates were subjected to Western blot analysis for GRP78 and c-MYC protein levels with GAPDH serving as loading control. Quantification of the relative protein levels of GRP78 and c-MYC, after normalization against GAPDH levels is shown in the graphs on the right (n=3). (E) Representative confocal immunofluorescence images of GRP78 (green) and c-MYC (red) staining in SCC25 cells after 48 h treatment with siCtrl or si78. The nuclei were stained by DAPI in blue. (Scale bars, 20 mm). Data are presented as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01 (Student's t-test).

Fig. 2

GRP78 small molecule inhibitor YUM70 suppresses c-MYC protein expression while upregulates its transcript levels in HNSCC. The HNSCC cells SCC25 (A), SCC15 (B), the cisplatin-resistant clonal lines cisR-2 (C), and cisR-3 (D) were treated with DMSO or YUM70 (10 μM) for 24 h. Whole cell lysates were subjected to Western blot analysis for c-MYC protein level with GAPDH serving as loading control. Total RNA was extracted and subjected to RT-qPCR to measure the mRNA levels of c-MYC with -β-actin serving as control. The quantitation of the relative c-MYC protein levels after normalization against GAPDH levels is shown in the graphs on the left (n=3) and the relative c-MYC mRNA level after normalization against β-actin mRNA is shown in the graphs on the right (n=5). (E) Representative confocal immunofluorescence images of c-MYC (red) staining in SCC25 cells after 24 h treatment with DMSO or YUM70 (10 μM). The nuclei were stained by DAPI in blue (Scale bars, 20 mm). Data are presented as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01 (Student's t-test).

Fig. 3

Effect of GRP78 small molecule inhibitors on the expression levels of eukaryotic translation initiation factors in HNSCC. (A) Schematic illustration of the assembly of various translation initiation factors on the 5′ cap region of mRNA, and translation inhibitor 4E-BP1 targeting eIF4E. (B) The HNSCC cells SCC25, SCC15, and the cisplatin-resistant clonal line cisR-3 were treated with DMSO or YUM70 (10 μM) for 24 h. Whole cell lysates were subjected to Western blot analysis for eIF4A, eIF4E, 4E-BP1, phosphorylated 4E-BP1 (p-4E-BP1), and eIF5A with GAPDH serving as loading control. Quantification of the relative protein levels of the indicated proteins, after normalization against GAPDH levels is shown in the graphs below (n=3). (C) Same as (B) except HA15 was used and the protein levels of c-MYC, 4E-BP1 and p-4E-BP1 were analyzed and quantified in the graphs below (n=3). Data are presented as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns denotes not significant (Student's t-test).

Fig. 4

YUM70 reduces c-MYC protein levels and induces apoptosis in 2D and 3D models of HNSCC. (A) Representative phase contrast microscopy images of SCC25 cells treated with DMSO or YUM70 (10 μM) for 48 h (Scale bar, 50 mm). (B) SCC25 cells were treated with DMSO or YUM70 (10 μM) for 24 h. Whole cell lysates were subjected to Western blot analysis for c-MYC and cleaved PARP (c-PARP) protein levels with GAPDH serving as loading control. (C) Same as (B) except SCC15, cisR-2, and cisR-3 cells were used. (D) Representative phase contrast microscopy image of cisR-2 spheroid culture (Scale bar, 200 mm). (E) cisR-2 or cisR-3 spheroids were treated with DMSO or YUM70 (10 μM) for 48 h. Whole cell lysates were subjected to Western blot for c-MYC and c-PARP protein levels with GAPDH serving as loading control.

Fig. 5

YUM70 reduces c-MYC expression and cell viability of c-MYC dependent human breast cancer. (A) Representative phase contrast microscopy images of MDA-MB-231 cells treated with DMSO or increasing doses of JQ1 as indicated for 48 h (Scale bars, 100 mm). (B) Same as (A) except the cells were harvested and whole cell lysates were subjected to Western blot analysis for c-MYC and cleaved PARP (c-PARP) protein levels with GAPDH serving as loading control. (C) Same as (A) except cell viability was measured by WST-1 assay at 24 or 48 h time points (n=3). (D) Representative phase contrast microscopy images of MDA-MB-231 cells treated with DMSO or YUM70 (10 μM) for 24 or 48 h (Scale bars, 50 mm). (E) Same as (D) except the cells were harvested and whole cell lysates were subjected to Western blot analysis for c-MYC and c-PARP protein levels with GAPDH serving as loading control. (F) Same as (D) except the cells were treated with increasing doses of YUM70 as indicated and cell viability was measured by WST-1 assay (n=3). (G) MDA-MB-231 cells were treated with either DMSO or YUM70 (10 μM) for 24 h and whole cell lysates were analyzed by Western blot for eIF4A, elF4E, 4E-BP1, phosphorylated-4E-BP1 (p-4E-BP1) and eIF5A protein levels with GAPDH serving as loading control. Quantification of the relative protein levels of the indicated proteins, after normalization against GAPDH levels is shown in the graphs on the right (n=3). (H) Same as (G) except the cells were treated with HA15 (10 μM) for 24 h and whole cell lysates were analyzed by Western blot for c-MYC, 4E-BP1 and p-4E-BP1 protein levels with GAPDH serving as loading control (n=3). Data are presented as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns denotes not significant (Student's t-test).

Fig. 6

YUM70 reduces c-MYC expression and cell viability in c-MYC dependent pancreatic cancer cells. (A) Representative phase contrast microscopy images of MIA PaCa-2 cells treated with DMSO or increasing doses of JQ1 as indicated for 48 h (Scale bars, 100 mm). (B) Same as (A) except the cells were harvested and whole cell lysates were subjected to Western blot analysis for c-MYC and cleaved PARP (c-PARP) protein levels with GAPDH serving as loading control. (C) Same as (A) except cell viability was measured by WST-1 assay at 24 or 48 h time points (n=3). (D) Representative phase contrast microscopy images of MIA PaCa-2 cells treated with DMSO or YUM70 (10 μM) for 24 or 48 h (Scale bars, 50 mm). (E) Same as (D) except the cells were harvested and whole cell lysates were subjected to Western blot analysis for c-MYC and c-PARP protein levels with GAPDH serving as loading control. (F) Same as (D) except the cells were treated with increasing doses of YUM70 as indicated and cell viability was measured by WST-1 assay (n=3). (G) MIA PaCa-2 cells were treated with control siRNA (siCtrl) or siRNA targeting GRP78 (si78) for 48 h. Whole cell lysates were subjected to Western blot analysis for GRP78 and c-MYC protein levels with GAPDH serving as loading control. Quantification of the relative protein levels of GRP78 and c-MYC, after normalization against GAPDH levels is shown in the graphs below (n=3). (H) MIA PaCa-2 cells were treated with either DMSO or YUM70 (10 μM) for 24 h and whole cell lysates were analyzed by Western blot for eIF4A, elF4E, 4E-BP1, phosphorylated-4E-BP1 (p-4E-BP1) and eIF5A protein levels with GAPDH serving as loading control. Quantification of the relative protein levels of the indicated proteins, after normalization against GAPDH levels is shown in the graphs on the right (n=3). Data are presented as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns denotes not significant (Student's t-test).

Fig. 7

YUM70 reduces c-MYC expression in 3D spheroid and xenograft models of pancreatic cancer. (A) Representative phase contrast microscopy images of MIA PaCa-2 spheroid culture treated with DMSO or YUM70 (10 μM) for 48 h (Scale bars, 200 mm). (B) Same as (A) except the cells were harvested and whole cell lysates were subjected to Western blot for c-MYC and c-PARP protein levels with GAPDH serving as loading control. (C) Schematic illustration of the MIA PaCa-2 xenograft experiment and treatment conditions. (D) MIA PaCa-2 xenograft tumor tissues were harvested from the mice at the end of the treatment and subjected to Western blot analysis for c-MYC protein level with GAPDH serving as loading control. The quantitation of the relative c-MYC protein levels after normalization against GAPDH levels is shown in the graphs on the right (n=4). (E) Proposed model of the mechanism by which GRP78 small molecule inhibitor YUM70 or HA15 suppresses MYC translational initiation through upregulation of the translation inhibitor 4E-BP1 which targets eIF4E, resulting in the displacement of eIF4G from the initiation complex, leading to reduced MYC translation and loss of cell viability. Data are presented as mean ± S.E.M. *p ≤ 0.05 (Student's t-test).