Depletion of Keratin8 affects tumorigenic potential of A431 cells via altering multiple signaling pathways
Keratins are the largest subgroup of intermediate filament proteins which are expressed in a tissue specific and differentiation state dependent manner. Apart from their mechanical functions, they have been shown to perform various regulatory roles. Most of these functions have been studied on keratin pair of 8/18 (K8/18) which is preferentially expressed in simple epithelia. The aberrant expression of this keratin pair has been correlated with increased invasiveness and poor prognosis of various squamous cell carcinomas (SCCs) including Skin SSCs. However the exact role it’s of aberrant expression in skin SCCs and associated mechanism remains elusive. To understand the role of K8/18 in skin SCC, ShRNA based stable K8 knockdown clones were generated in skin epidermoid carcinoma derived A431 cells which resulted in the decreased tumorigenic potential of these cells. Next, to decipher the molecular basis behind K8 mediated regulation of tumorigenic potential of A431 cells, TMT based total quantitative proteomics was performed for vector control and K8 knockdown clones (K8KD1/K8KD2). There were a total of 2952 proteins detected in the TMT based total quantitative proteomics, out of which, K8KD1 clone showed 140 proteins to be differentially expressed, whereas K8KD2 clone showed 119 proteins to be differentially expressed in comparison to vector control cells. Further, functional analysis of our data showed involvement of cell death and survival together with cellular movement pathway to be affected upon K8 knockdown. Altogether, the present data potentiates the role of K8 in neoplastic progression of skin SCC, together with the possible mechanism underlying the same.
Sample Processing Protocol
Cell lysates were prepared in lysis buffer TEABC lysis Buffer. The cell lysates were sonicated and centrifuged at 12,000 rpm for 10 minutes. The protein concentration was estimated using Bicinchoninic acid assay (BCA). 8mg of proteins from each cell type were reduced by 5mM DTT and alkylated using 10mM iodoacetamide for 10 minutes at room temperature in the dark. We employed filter aided sample preparation (FASP) protocol to remove SDS. After SDS removal again protein estimation was carried out. 1.4 mg of proteins from each cell types were subjected to protein using TPCK treated trypsin at 37°C for 12-16hours. Peptides from each cell type were labelled using 10plex-TMT labelling method as per manufacturer’s instruction. Peptides derived from A431 parental cell line were labelled with TMT tag 126 and 127N, Vector control cells (VC) with TMT tag 128N and 128C, K8 knockdown clone-2 (K8-KD2) with TMT tag 129N and K8 knockdown clone-1 (K8-KD1) with TMT tag 130C and 131. After labelling all the peptides were pooled and subjected to basic reverse phase liquid chromatography. The peptides were analyzed on Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Electron, Bremen, Germany) interfaced with Easy-nLC 1000 liquid chromatography system (Thermo Scientific, Odense, Denmark). The peptide digests were reconstituted in 0.1% formic acid and loaded onto trap column (75 µm x 2 cm) packed in-house with Magic C18 AQ (Michrom Bioresources, Inc., Auburn, CA, USA). Peptides were resolved on an analytical column (75 µm x 20 cm) at a flow rate of 350 nL/min using a linear gradient of 10-35% solvent B (0.1% formic acid in 95% acetonitrile) over 80 minutes. Data dependent acquisition with full scans in 350-1700 m/z range was carried out using an Orbitrap mass analyzer at a mass resolution of 120,000 at 400 m/z. Fifteen most intense precursor ions from a survey scan were selected for MS/MS fragmented using HCD fragmentation with 32% normalized collision energy and detected at a mass resolution of 30,000 at 400 m/z. Dynamic exclusion was set for 30 seconds with a 10 ppm mass window. Internal calibration was carried out using lock mass option (m/z 445.1200025) from ambient air.
Data Processing Protocol
The mass spectrometry derived data was searched against “Human RefSeq protein database” (Version 65, containing 36211 protein entries with common contaminants added) using “SEQUEST” and “Mascot” search algorithms through “Proteome Discoverer” platform (version 220.127.116.11, Thermo Scientific). The search parameters for both algorithms included: carbamidomethylation of cysteine and TMT 10-plex (C229.163) modification at N-terminus of peptide and lysine as fixed modifications. MS/MS spectra were searched with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05Da. Trypsin was specified as protease and a maximum of two missed cleavages were allowed. The data were searched against decoy database and the false discovery rate was set to 1% at the PSM level. Peptide with ratio ≥1.5-fold cut-off were considered for further analysis.
Tiwari R, Sahu I, Soni BL, Sathe GJ, Thapa P, Patel P, Sinha S, Vadivel CK, Patel S, Jamghare SN, Oak S, Thorat R, Gowda H, Vaidya MM. Depletion of Keratin 8/18 modulate oncogenic potential by governing multiple signaling pathways. FEBS J. 2018 PubMed: 29427328