Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress.

Valosin-containing protein (VCP), also called p97, is a AAA+ ATPase that has been shown to be involved in endoplasmic reticulum-associated protein degradation (ERAD), mitochondria quality control and vesicle transport. We and others have previously found that disruption of VCP is sufficient to cause endoplasmic reticulum (ER) stress. We observed that induction of ER stress either following siRNA mediated loss of VCP or inhibition of VCP with eeyarestatin I potently activates an EMT-like state in cells. Interestingly, both ER stress and EMT are reversible events. Further, brief treatment of cells with eeyarestatin I increases EMT markers, and migratory and invasive properties of lung cancer cells. By examining primary lung adenocarcinoma patient samples we find that the VCP locus is heterozygously lost in nearly half of lung adenocarcinomas and VCP protein expression is decreased in nearly all primary lung tumors. Further, primary lung adenocarcinomas have increased ER stress and EMT markers. These observations have potential clinical relevance because increased ER stress and EMT markers are known to contribute to chemoresistance and poor survival of patients with lung adenocarcinoma.

2 time following transfection, cells were reseeded in 6 well plates in triplicate for each transfection. Cells were allowed to grow on 6 well plates for 10 days. Cells were supplemented with fresh media after every 2-3 days. After 10 days, colonies formed were washed once with PBS, fixed with 70% ethanol and stained with 0.25% coomassie brilliant blue stain R (#B 7920 Sigma-Aldrich, Inc. St. Louis, MO. USA) and photographed.

RNA Extraction, cDNA Synthesis, and RT-PCR
After 48 hrs of treatment either with vehicle alone or with EerI and further 48 hrs without EerI in medium and similarly, after 72 hrs of transfection of A549 and H358 cells either with nontargeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 and siVCP2), cells were harvested and total RNA was purified using TRI reagent (#9424) (Sigma-Aldrich, Inc. St. Louis, MO. USA), according to the manufacturer's protocol. The quality of the isolated RNA was assessed using the Bioanalyzer 2100 (Agilent Technologies, Inc., Wilmington, DE) and quantification was performed using a Nanodrop 1000 (Thermo Scientific, Wilmington, DE). cDNA was synthesized with High Capacity RNA-to-cDNA Kit from Applied Biosystems # 4387406 (Applied Biosystem, Foster City, USA). The PCR amplification was performed by using One Taq TM Hot Start 2X Master Mix #M0484S (New England Biolabs, Ipswich, MA), with 0.2 µM of each primer, 10 µl of 2X master mix and 1 µl of cDNA template, in a final reaction volume of 20 µl using the following cycle parameters: enzyme activation at 94 0 C for 30 s; 35 cycles of 94 0 C for 15 s, 60 0 C for 30 s and 68 0 C for 1 min, and 5 min of final extension at 68 0 C. PCR products were directly analyzed by separation on 3% agarose gels. All experiments were repeated three times using three independent preparations of cDNA.

Drug treatments
A549 and H358 cells either treated with vehicle alone or with indicated concentrations of Eeyarestatin (EerI) (Cayman) for 48 hrs. Cells were then trypsinized and used for further studies or then harvested and total lysates were used for expression studies. In case of Akt inhibitor BEZ235 (Cayman) treatment, cells were exposed to either vehicle alone or Eeyarestatin I (20µM) for 48 hrs and treated with BEZ235 (1µM) for 24 hrs before harvesting. For Src kinase inhibitor treatment, cells were pretreated with PP2 (Cayman) (20µM) and then cotreated with 4 either vehicle alone or Eeyarestatin I (20µM) for 48 hrs. Cells were then harvested and total lysates were used for expression studies.
Cell migration assay or Wound healing assay A549 cells were plated 100mm plate and exposed to either vehicle alone or EerI for 48 hrs. Cells were washed with PBS, trypsinized, counted and replated in triplicate in 6-well plate. Next day, wound were made using the pipette tip following replacement with fresh media. Cells were examined successively after 24 hrs and 48 hrs of wound formation and photographed. Average of percentage wound closure of randomly selected three areas was calculated by using ImageJ (NIH, USA) software. were transfected with either non-targeting (siNT) siRNA or siRNA targeting VCP (siVCP). Cells were exposed to either vehicle alone or CH for indicated time before total 72 hrs of transfection.
Cells were harvested at the indicated time points. Representative western blot analysis showing expression of proteins involved in cell cycle regulation.