ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

Abstract

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DNp63a associated with its increased phosphorylated status. Since DNp63a usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DNp63a in HNSCC cells upon cisplatin exposure. We found that DNp63a is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DNp63a in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DNp63a residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DNp63a degradation. Finally, we demonstrated that the DNp63a protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DNp63a following DNA damage.

Phospho-ΔNp63α is a key regulator of the cisplatin-induced microRNAome in cancer cells

“…[18][19][20] We previously observed that the head and neck squamous cell carcinoma (HNSCC) cells exposed to CIS displayed a dramatic downregulation of DNp63a through an ATMdependent phosphorylation mechanism. 21,22 We also showed that phospho (p)-DNp63a is critical for the transcriptional regulation of downstream mRNAs in HNSCC cells. 21,22 In the current study, we present evidence that p-DNp63a regulates miRNA expression in CIS-treated HNSCC cells through both transcriptional and post-transcriptional mechanisms.…”

Phospho-ΔNp63α/microRNA network modulates epigenetic regulatory enzymes in squamous cell carcinomas

“…[35][36][37][38][39] Using knock-in technology, we generated SCC-11 cells, which have been shown to produce wild-type ΔNp63α, and SCC-11M cells that exclusively express ΔNp63α-S385G mutant protein, with an altered ability to be phosphorylated by ATM kinase. 34 By global analysis of microRNA expression, we previously showed that cisplatin exposure led to a downregulation of 28 microRNAs (e.g., miR-519a-3p, miR-181a-5p, miR-374a-5p, miR-98-5p, miR-29c-3p, miR-22-3p, miR-34c-3p, miR-206, miR-429, miR-339-3p, miR-203a, miR-25-3p, miR-155-5p, and miR-148a-3p) by −5.18 to −19.27-fold, and upregulation of 15 microRNAs (e.g., miR-382-3p, miR-485-5p, miR-574-5p, miR92b-3p, miR-297, miR-185-5p, miR-885-3p, miR-194-5p, and miR-630) by 3.95-to 7.46-fold in SCC-11 cells compared with SCC-11M cells upon cisplatin exposure. [36][37][38][39] We further showed that cisplatin exposure altered microRNA expression in SCC-11 cells, resulting in downregulation of 7 microRNAs (e.g., miR-519-a-3p, miR-181a-5p, miR-374a-5p, miR-29c-3p, miR-98-5p, miR-22-3p, and miR-34c-3p, from −1.72 to −3.77-fold), and upregulation of 7 microRNAs (miR-382-3p, miR-485-5p, miR-574-5p, miR-297, miR-194-5p, miR-885-3p, and miR-630, from 2.08-to 4.98-fold), as shown in references 36-39.…”

“…33 We have previously shown that the SCC cells exposed to cisplatin treatment displayed a dramatic downregulation of ΔNp63α via an ATM-dependent phosphorylation mechanism. 34 We have also shown that the phosphorylated (p)-ΔNp63α protein is critical for the transcriptional regulation of downstream mRNAs and microRNAs in SCC cells upon cisplatin exposure. 35,36 Moreover, we have reported that p-ΔNp63α regulates microRNA expression in cisplatin-treated SCC cells through both transcriptional and post-transcriptional mechanisms.…”

“…The custom rabbit polyclonal antibody against phosphorylated peptide encompassing the ΔNp63α protein sequence (ATM motif, NKLPSV-pS-QLINPQQ, residues 379-392) was also used. 34,35 Transfection with microRNA mimics The following individual human mirVana ® microRNA mimics (hsa-miR-297, hsa-miR-92b-3p, and hsa-miR-485-5p) were purchased from Ambion/Life Technologies. Cells in a 6-well plate were transfected with 100 pmol of the mimic or scrambled RNA in 500 μl serum-free media with 5 μl of Lipofectamine-2000 reagent (Invitrogen) for 32 h. Each experiment was performed independently 3 times and in triplicate.…”

“…[34][35][36][37][38][39]100 Stable SCC cell lines expressing wild-type ΔNp63α (SCC-11) or ΔNp63α-S385G (SCC-11M) were generated using Flp-In technology. 34 We also used SCC-25 cells (expressing mutated TP53 [R209] and CDKN2A, expressing ΔNp63α) and SCC-25CP cells (with a spontaneously acquired cisplatin resistance) derived from the primary tongue SCC, as previously reviewed. 39,44,101 Cells were maintained in a 1:1 mixture of Dulbecco modified Eagle medium and Ham F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate and supplemented with 400 ng/ml hydrocortisone and 10% fetal bovine serum.…”