Figure 1.
Elevated VCP protein levels and proteostasis-imbalance in NSCLC.
(a) We examined the expression of VCP in adeno(AD)- and squamous(SQ)-NSCLSC as compared to normal tissue and observed a significant increase (p<0.001) in VCP expression and ubiquitin accumulation in both NSCLCs (n = 4, White bar = 100 µm). (b) We confirmed that VCP and ubiquitinated proteins are co-localized in peri-nuclear aggregates of these tissues (n = 4, Black bar = 100 µm & Red bar = 10 µm). Elevated VCP protein expression and ubiquitin accumulation in NSCLC indicates proteostasis-imbalance.
Figure 2.
VCP regulates NSCLC proliferation, apoptosis and cell division.
(a) H1299 cells were seeded on a 96-well plate and either transfected with pSM2 control vector or VCP-shRNA (for 16 hrs) or treated with EerI (10 µM) or DMSO (vehicle) for 24 hrs. Promega Aqueous One MTS reagent was added to each well, 2 hours before stopping the experiment and VERSAmax microplate reader was used to quantify the MTS activity (n = 4) at indicated time points. Our data shows a significant (p<0.01) decrease in cell proliferation by VCP inhibition. (b) H1299 cells were seeded on a black-bottomed 96-well plate and either transfected with pSM2 control vector or VCP-shRNA or treated with EerI or DMSO. After 48 (VCP-shRNA) or 24 (EerI) hours, caspase-3/7 activity was quantified using the luminescence substrate (Promega). Our data shows an increase in caspase-3/7 activity after transient VCP knockdown (* p<0.05) and a very significant increase (two-fold) in caspase-3/7 activity after treatment with functional inhibitor of VCP, EerI (*** p<0.00001). Next, (c) H1299 cells were either transfected with VCP-shRNA2 and pSM2 control vector or (d) treated with EerI or DMSO. Cells were then fixed in 70% ethanol followed by propidium iodide (10 µg/ml) staining for 1 hour. The profiles of DNA content (measured by a FACScan flow cytometer) for cell-cycle distributions are shown. Our data, summarized in the bar graph (bottom of each panel), shows that VCP knockdown induced G0/G1 arrest (*** p<0.001) and reduced the number of cells undergoing DNA replication (S-phase; ** p<0.01). Inhibition of VCP's function by EerI not only induced G0/G1 arrest (** p<0.01) and decreased DNA replication (** p<0.01) but also significantly reduced the number of cells undergoing mitosis (G2/M-phase; * p<0.05). VCP inhibition controls the tumorigenic capacity of NSCLC by slowing proliferation and division while simultaneously inducing apoptosis.
Figure 3.
VCP inhibition retards the migration, invasion and growth of NSCLC.
H1299 cells were either (a) transfected with pSM2 control vector or VCP-shRNA or (b) treated with EerI or DMSO, followed by scratch assay to quantify changes in cell migration. Briefly, a scratch was made across the center of the confluent monolayer using a 10 µl pipette tip. The cells were allowed to migrate and were monitored at the indicated time points using a Nikon light microscope and Infinity Capture Software. The representative images show that both VCP knockdown and inhibition visibly slowed H1299 migration into the scratch by 12 and 24 hours (top panels). Changes in the average width of the scratch were calculated using Infinity Analyze Software (mean ± SEM; n = 3–4). Quantitative analysis show that both VCP knockdown and functional inhibition significantly retarded cell migration at 12 and 24 hours (bottom panels, * p<0.05; ** p<0.0005). (c) H1299 cells were transfected with pSM2 or VCPshRNA2 for 24 hours and transferred to transwell inserts (BD, 0.4 µm pores) coated with 200 µg Matrigel basement matrix for additional incubation for 24 hours. Following incubation, cells that had migrated to the bottom of the membrane were stained with trypan blue solution and the central field of each insert was visualized using a Nikon light microscope (n = 4, mean ± SEM given). The data shows that VCP inhibition significantly reduced the number of invading cells as compared to the controls. (d) H1299 cells (4.0×105 cells/well) were suspended in 2 mL of serum-containing medium with agarose (0.3%) and immediately plated over a layer of solidified agarose (2-mL, 0.6%) on a 6-well plate (n = 3). No cells (Blank) and untreated H1299 cells were used as controls. Fresh media containing either EerI (10 µM) or DMSO (vehicle) was added on top. After 48 hrs, the images were captured and analyzed using the Adobe Photoshop Software. Representative images and densitometry data (mean ± SEM) shows the significant decrease in number of colonies and H1299 cell invasion in agarose by EerI treatment as compared to the DMSO treated controls. VCP mediates NSCLC migration, invasion and growth that suggest its critical role in tumor cell progression and metastasis.
Figure 4.
VCP regulates protein levels of p53, NFκB and other cancer-related proteins.
(a) We observed the accumulation of ubiquitinated proteins, induction of VCP, Nrf2, SIRT1 (Sirtuin 1, stress regulator), and NFκB, and suppression of p53 in NSCLC cell lines (H1299 and H1944) as compared to control (Beas2b and HBE) cells indicating towards proteostasis-imbalance. β-actin antibody was used as an equal-loading control. NFκB, p53, Nrf2 and SIRT1 are known to be involved in tumor metastasis, proliferation, apoptosis and inflammatory-oxidative stress responses. (b) VCP overexpression (VCP-myc) in H1299 cells shows an increase in NFκB protein levels. (c) H1944 cells transfected with VCP shRNA (lane 4–6) show a significant increase in p53 protein levels and decrease in NFκB, as compared to pSM2 control (lanes 1–3). VCP immunoblot verifies VCP inhibition while β-actin shows equal loading. (d) Co-immunoprecipitation (IP) of p53 with VCP antibody verifies the novel protein-protein interaction. IP lacking anti-VCP antibody was used as a negative control. Regulation of oncogene and tumor suppressor by VCP provides a potential mechanism for its critical role in tumor progression and metastasis.
Figure 5.
Inhibition of VCP's function by EerI reduces tumor growth in NSCLC-xenograft model.
(a) Tumors were established in athymic nude mice by subcutaneous (s.c.) injection of H1299 (8×106) cells complexed with Matrigel into the upper left flank. Mice were randomized into two groups (n = 5) and treated with EerI (50 µg, 2 doses at indicated times as on the scale) or DMSO vehicle. (b) Primary tumor growth was measured volumetrically over time and statistical analysis of data is shown as mean ± SEM. EerI treatment significantly reduced tumor volume as compared to the DMSO vehicle control ** p<0.01). (c) Representative images captured on Day 8 illustrate the difference in tumor volumes between the two groups. VCP can be therapeutically targeted using EerI or other potent VCP-inhibitor to control NCLSC tumor growth and/or progression.
Figure 6.
Schematic summarizing the functional role of VCP in NSCLC pathogenesis and progression.
Our data suggests that elevated VCP expression and subsequent accumulation of ubiquitinated proteins (proteostasis-imbalance) de-regulates NSCLC proliferation, cell cycle, apoptosis and migration via NFκB and p53 pathways resulting in increased tumor- genesis and metastasis. We predict that increased retrograde translocation rates results in the accumulation of ubiquitinated aggregates that leads to chronic activation of VCP. This chronic VCP induction may lead to the pathological expression of key cancer-related proteins, including inhibition of tumor suppressor p53 and activation of key metastasis-protein NFκB, as seen in our study. We propose that inhibition of VCP expression and/or function may control the progression and aggressiveness of NSCLC by rescuing vital cellular pathways from pathogenic activation.