DEPTOR is linked to a TORC1-p21 survival proliferation pathway in multiple myeloma cells

Abstract

We investigated the mechanism by which gene silencing of the mTOR inhibitor, DEPTOR, induces cytoreductive effects on multiple myeloma (MM) cells. DEPTOR knockdown resulted in anti-MM effects in several MM cell lines. Using an inducible shRNA to silence DEPTOR, 8226 MM cells underwent TORC1 activation, downregulation of AKT/SGK activity, apoptosis, cell cycle arrest and senescence. These latter cytotoxic effects were prevented by TORC1 paralysis (Raptor knockdown) but not by over-expression of AKT activity. In addition, DEPTOR knockdown-induced MM death was not associated with activation of the unfolded protein response, suggesting that enhanced ER stress did not play a role. In contrast, DEPTOR knockdown in 8226 cells induced p21 expression, independent of p53, and p21 knockdown prevented all of the cytotoxic effects following DEPTOR silencing. DEPTOR silencing resulted in p21 upregulation in additional MM cell lines. Furthermore, DEPTOR silencing in a murine xenograft model resulted in anti-MM effects associated with p21 upregulation. DEPTOR knockdown also resulted in a decreased expression of p21-targeting miRNAs and transfection of miRNA mimics prevented p21 upregulation and apoptosis following DEPTOR silencing. Use of a shRNA-resistant DEPTOR construct ruled out off-target effects of the shRNA. These results indicate that DEPTOR regulates growth and survival of MM cells via a TORC1/p21 pathway and suggest an involvement of p21-targeted miRNAs.

Keywords: AKT; DEPTOR; ER stress; Multiple Myeloma; mTORC1; p21.

Figure 1. DEPTOR knockdown induces Apoptosis, G1/S cell cycle arrest & senescence in MM cells

A). Cell lines acutely infected with control (scramble) or two separate DEPTOR shRNAs followed by Western blot. Phospho-P70/total P70 ratio shown under the gel was determined by densitometry and is mean of 3 separate experiments; B) % apoptosis 3 or 4 days after lentivirus infection with control (scr) or shRNA to DEPTOR (sh1, sh2). C) 8226 transfected with dox-inducible control shRNA (Luc or GFP) or shRNA targeting 2 sequences of DEPTOR (DEPTOR-1/DEPTOR-2); Dox added to induce shRNAs and immunoblot 72 hrs later for DEPTOR expression or PARP cleavage (arrow); D) Immunoblot assay performed in 8226 cells at 1, 2 or 3 days after adding dox; E) MTT assay over time after adding dox to induce shRNA; F) Apoptosis assay at 72 hrs (flow analysis of activated caspase 3) in control (shLuc) or DEPTOR knockdown cells (shDEP) +/− dox. ZVAD added at 50 or 100 uM. G) G1 or S distribution in transfected 8226+/− dox. H) Senescence assay (SA-beta gal staining (arrows)) in DEPTOR knockdown cells after adding dox; I) Quantification of senescence. All data in figs 1B, 1E, 1F, 1G and 1I represent means+/−SD, n=3).

Figure 2. Molecular effects of DEPTOR knockdown in 8226 cells

A) Control (Luc) or DEPTOR knocked down cells +/− dox, followed by Western blot; B)Extract from same cells as in ‘A’ immunoblotted for NDRG-1 or phosphorylated AKT. AKT also immunoprecipitated from extracts and tested for kinase activity against GSK (bottom).

Figure 3. Apoptosis induced by DEPTOR knockdown is TORC1-dependent but independent of AKT inhibition

A) Inducible shRNA cell line infected with shcontrol (scramble) or shRNA targeting RAPTOR followed by Western blot; B)Same cells as in ‘A’ treated +/− dox to induce DEPTOR silencing, followed by Western Blot; C)Same cells as in ‘A’ or ‘B’, treated +/− dox, followed by Western blot for phospho-AKT or in AKT vitro kinase activity against GSK (bottom); D) DEPTOR knockdown-induced apoptosis is prevented by RAPTOR knockdown (mean+/−SD, n=4, RAPTOR knockdown significantly (p<0.05) inhibited dox-induced apoptosis); E) Effects of ectopic expression of wild type (WT) AKT or phosphomimetic AKT (AKT S473D); F) Same cells as in ‘E’ treated +/− dox to silence DEPTOR, followed by Western blot. G) DEPTOR knockdown induction of apoptosis is unaffected by ectopic over-expression of AKT (mean+/−SD, n=3) although bortezomib-induced apoptosis is significantly inhibited (p<0.05).

Figure 4. DEPTOR knockdown-induced Apoptosis is not due to heightened ER stress

A) shRNA-transfected 8226 cells treated +/− dox followed by immunoblot; B) DEPTOR shRNA-transfected 8226 cells treated +/− bortezomib, followed by Western blot; C) shRNA-transfected 8226 cells treated +/−thapsigargin (Tg in uM) or dox to induce DEPTOR knockdown, followed by immunoblot assay; D) DEPTOR shRNA-transfected 8226 cells treated +/− dox and +/− increasing concentrations (uM) of JNK inhibitor, followed by immunoblot; E)Same cells as in ‘D’ treated +/− bortezomib +/− JNK inhibitor; F) DEPTOR shRNA-transfected cells treated with dox or bortezomib and increasing concentrations of SP600125 JNK inhibitor, followed by apoptosis assay. Data are means+/− SD, n=3; *=different from control (no inhibitor), p<0.05.

Figure 5. DEPTOR knockdown-induces P21 and P27 in a P53-independent fashion

A) Transfected cells treated +/− dox followed by immunoblot. B) Inducible shDEPTOR cells ectopically expressing scramble or RAPTOR shRNAS treated +/− dox followed by immunoblot; C) Inducible shDEPTOR cells ectopically expressing EV or AKT(WT) or phosphomimetic AKT treated+/− dox followed by immunoblot; D) qt-PCR for p21 RNA expression following infection with control (shLuc) or DEPTOR shRNA and treated+/− dox (mean+/− SD, n=3). E) Acute infection with shRNA scramble (control) or DEPTOR shRNA in OPM-2 cells followed by immunoblot; F) Mice (8/group) challenged with SQ injection of inducible 8226 cells and, when tumor size reached 400 mm³, mice were randomized to receive dox in water or no dox. Tumor size is mean+/−SDs. G) Western blot of tumor protein extracted from 2 separate mice from each group.

Figure 6. P21 upregulation induced by DEPTOR knockdown mediates Apoptosis and cell cycle arrest

A)P21 expression in inducible 8226 cells transduced with scramble shRNAs or shRNAs targeting p21; B)MTT assay showing growth (over time) in same cells as in ‘A’ treated +/− dox. Data are means+/−SD, n=3; C) Apoptosis assay in transfected cells treated+/− dox. Data are means+/−SD, n=3. P21 silencing significantly (p<0.05) inhibited dox-induced apoptosis; D) Cell cycle analysis in same cells. Data are means of 3 separate experiments. E) Senescence assay in same cells. Data are means+/−SD, n=3; *=different by p<0.05.

Figure 7. Role of miRNAs

A) qt-PCR assessment in 2 MM cell lines of expression of 3 miRNAs of the 106b family following DEPTOR silencing by acute infection with 2 independent shRNAs (black bars=8226; white bars=OPM-2); B) Dox-inducible 8226 cells (upper panel) not treated (parental), treated with Lipofectamine 2000 (LP2000) alone, transfected with control mimics (control) or mimics of all 3 miRNAs (mimics). Cells then exposed to dox to silence DEPTOR followed by immunoblot. OPM-2 cells (lower panel) similarly treated followed by acute infection with control or shRNA targeting DEPTOR; C) The experiments in ‘B’ were repeated 3x and p21/mTOR or p27/mTOR ratios determined by densitometry. Data show mean+/−SD.

Figure 8. Use of shRNA-resistant DEPTOR

A) 8226 MM cells transfected with empty vector (EV), mutant DEPTOR (resistant) or wild type (WT) DEPTOR and either treated with or without shRNA targeting DEPTOR, followed by immunoblot; B) % apoptosis (mean+/− SD, n=3) following DEPTOR knockdown (+/−shRNA) in EV, mutant-DEPTOR (resistant) or wild type DEPTOR-transfected 8226 cells. C) qt-PCR quantification of relative amounts of miRNA 106a, 93 and 20b in MM cells transfected as in ‘A’ (EV=empty vector; WT=wild type DEPTOR; Mu=mutant (resistant) DEPTOR). Data are means+/−SD, n=3. D) Model of events following DEPTOR knockdown in MM cells.