The Hydroxyquinoline Analogue YUM70 Inhibits GRP78 to Induce ER Stress-Mediated Apoptosis in Pancreatic Cancer

Abstract

GRP78 (glucose-regulated protein, 78 kDa) is a key regulator of endoplasmic reticulum (ER) stress signaling. Cancer cells are highly proliferative and have high demand for protein synthesis and folding, which results in significant stress on the ER. To respond to ER stress and maintain cellular homeostasis, cells activate the unfolded protein response (UPR) that promotes either survival or apoptotic death. Cancer cells utilize the UPR to promote survival and growth. In this study, we describe the discovery of a series of novel hydroxyquinoline GRP78 inhibitors. A representative analogue, YUM70, inhibited pancreatic cancer cell growth in vitro and showed in vivo efficacy in a pancreatic cancer xenograft model with no toxicity to normal tissues. YUM70 directly bound GRP78 and inactivated its function, resulting in ER stress-mediated apoptosis. A YUM70 analogue conjugated with BODIPY showed colocalization of the compound with GRP78 in the ER. Moreover, a YUM70-PROTAC (proteolysis targeting chimera) was synthesized to force degradation of GRP78 in pancreatic cancer cells. YUM70 showed a strong synergistic cytotoxicity with topotecan and vorinostat. Together, our study demonstrates that YUM70 is a novel inducer of ER stress, with preclinical efficacy as a monotherapy or in combination with topoisomerase and HDAC inhibitors in pancreatic cancer. SIGNIFICANCE: This study identifies a novel ER stress inducer that binds GRP78 and inhibits pancreatic cancer cell growth in vitro and in vivo, demonstrating its potential as a therapeutic agent for pancreatic cancer.

Figure 1.. YUM70 is cytotoxic to pancreatic cancer cell lines.

A. Chemical structure of YUM70. B. Cytotoxicity of YUM70 in a panel of pancreatic cancer cell lines and normal pancreatic tissue-derived cells (HPNE) measured by the MTT assay. IC₅₀ presented as mean ± SD of three independent experiments performed in duplicate. C. YUM70 dose-dependently decreased PANC-1 and UM59 cell proliferation in 3D-culture systems. D. Quantification of cell viability of 3D spheroids was performed with CellTiter-Glo® 3D cell viability assay. Data are presented as mean ± SD of three or more spheroids from three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001

Figure 2.. YUM70 upregulates UPR target proteins and induces ER stress and apoptosis.

A. Expressions of DDIT3 (CHOP) and HSPA5 (GRP78) are upregulated by YUM70 treatment (Bru-seq data). B. GSEA analysis of YUM70 treatment reveals enrichment of ER stress and apoptosis pathway. C–D. Lysates of MIA PaCa-2 and PANC-1 cells treated with YUM70 at indicated doses and times were immunoblotted with indicated antibodies. A representative data set from three independent experiments is shown. E-F. Relative expression (fold change) of FAM129A, GRP78, and CHOP normalized to GAPDH/Actin in MIA PaCa-2 and PANC-1 cells were calculated from the band intensity of three independent experiments and are presented as mean fold change at indicated time (hr). G-H. Representative immunoblot and relative expression (mean fold change) of the above stated proteins in BxPC-3 cells are presented. DMSO control indicated as Ctrl. Error bars represent standard deviation. The band intensity was quantified in Image Studio Ver 3.1 software. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. YUM70 upregulates proteins involved in the apoptotic pathway.

A-B. Lysates of MIA PaCa-2 and PANC-1 cells were immunoblotted with indicated antibodies. A representative experiment out of three is shown. The actin blot used in Figure 3A is the same as in Figure 2C. C-D. Caspase 3/7 activity levels in MIAPaCa-2 and PANC-1 cells, following treatment with YUM70. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. E. YUM70 dose-dependently causes apoptosis in MIA PaCa-2 and PANC-1 cells. Top, cells in the bottom left quadrant of each panel (Annexin V–negative, PI-negative) are viable, whereas cells in the bottom right quadrant (Annexin V–positive, PI-negative) are in an early stage of apoptosis, and cells in the top right quadrant (Annexin V–positive, PI-positive) are in a late stage of apoptosis/necrosis. Bottom, the percentage of apoptotic cells is shown in a histogram. A representative image of three independent experiments is shown.

Figure 4.. YUM70 targets GRP78.

Apparent melting temperature (Tm) derived from thermal shift assay and the corresponding thermal shift were determined at various concentrations of A. YUM70 B. VER and C. YUM117. 1% DMSO was used as control. Data are presented as mean ± SD. *p < 0.01, **p < 0.001, ***p < 0.0001. Numbers in red are thermal shift at respective concentrations. D. CETSA melt curves for GRP78 in PANC-1 cell lysates treated with YUM70 (100 μM). 1% DMSO was used as control. Data are presented as mean ± SD of three independent experiments. E. Inhibition of GRP78 enzymatic activity in the presence or absence of YUM70 and VER. The results are presented as mean % inhibition of GRP78 of three independent experiments. Error bars represent standard deviation. F. PERK and IRE1α were immunoprecipitated overnight from PANC-1 cell lysates in the presence of DMSO, YUM70 or YUM117. Western blotting was performed with indicated antibodies. A representative experiment out of three is shown. G. Chemical structure of YUM401 and YUM488 (BODIPY-conjugated YUM401). H. YUM488 co-localizes with GRP78 in the ER. Immunofluorescence image of MIA PaCa-2 treated with tunicamycin for 2 hr followed by 15 μM YUM488 treatment for 16 hr. ER was labeled with ER-Tracker (detection wavelength 566–626 nm), GRP78 was labeled with antibody (detection wavelength 656–700 nm), and YUM488 was detected at wavelength 497–564 nm.

Figure 5.. YUM70 inhibits pancreatic tumor growth in vivo.

A. Tumor growth curves of MIA PaCa-2 tumor-bearing nude mice treated with vehicle (n = 5) or YUM70 (n = 5). Data are shown as mean tumor volumes (error bars, SEM). A significant reduction in tumor volumes was observed upon YUM70 treatment (*p < 0.05). B. Evaluation of mouse weights during the xenograft experiment. Error bars indicate mean ± SEM. C. Ki67 immunohistochemistry staining in tumor sections. D. Percent of Ki67 positive cells were calculated as the fraction of Ki67 positive cells compared to the total number of cells in the field × 100. (n = 10; five fields of view from two tumors per group). Graphical data is presented as Mean ± SD, ***p < 0.0001. E. Lysate from two tumors per group was blotted for FAM129A, GRP78, CHOP, cleaved caspase 3. ‘M’ is the molecular weight marker. Normalized relative densities computed using ImageJ (NIH) are shown above. F. YUM70 did not show systemic toxicity in vivo. Representative micrographs of hematoxylin and eosin (H&E)-stained organ tissue sections. Images were taken with an Olympus IX83 inverted microscope at 20X magnification. G. Mechanism of YUM70 mediated cell death. YUM70 is cytotoxic to pancreatic cancer cells via a mechanism involving the allosteric inhibition of GRP78 activity, leading to an increase in ER stress and induction of protein levels of UPR target genes. As a result, YUM70 treatment causes prolonged ER stress-mediated apoptosis.

Figure 6.. Synergistic effect of YUM70 in combination with topotecan and vorinostat.

MIA PaCa-2 cells were treated with YUM70 with or without topotecan (Topo) and vorinostat (SAHA), at stated concentrations and kept in culture until colonies were observed in DMSO treated control. A. A representative image is shown (one concentration). B and C. The number of colonies was quantified using Image Studio ver3.1 software from three independent experiments (more than one concentration). Graphical data is presented as mean ± SD, *p < 0.05, **p < 0.01. The p-value of the combination was calculated and compared to YUM70 alone. D and E. The combined effect was calculated using CompuSyn software. CI <1 is defined as synergism. F. Combination regimen causes apoptosis in MIA PaCa-2 and PANC-1 cells. Top, cells in the bottom left quadrant of each panel (Annexin V–negative, PI-negative) are viable, whereas cells in the bottom right quadrant (Annexin V–positive, PI-negative) are in the early stage of apoptosis, and cells in the top right quadrant (Annexin V–positive, PI-positive) are in the late stage of apoptosis/necrosis. Bottom, the percentage of apoptotic cells is shown in a histogram. A representative image of three independent experiments is shown.