Deptor transcriptionally regulates endoplasmic reticulum homeostasis in multiple myeloma cells

Abstract

Multiple myeloma (MM) is a malignant disorder of plasma cells characterized by active production and secretion of monoclonal immunoglobulins (IgG), thus rendering cells prone to endoplasmic reticulum (ER) stress. For this reason, MM cell survival requires to maintain ER homeostasis at basal levels. Deptor is an mTOR binding protein, belonging to the mTORC1 and mTORC2 complexes. It was reported that Deptor is overexpressed in MM cells where it inhibits mTOR kinase activity and promotes cell survival by activating Akt signaling. Here we identify Deptor as a nuclear protein, able to bind DNA and regulate transcription in MM cells. In particular, we found that Deptor plays an important role in the maintenance of the ER network, sustaining the expression of several genes involved in this pathway. In agreement with this, Deptor depletion induces ER stress and synergizes the effect of the proteasome inhibitor bortezomib (Bz) in MM cells. These findings provide important new insights in the ER stress control in MM cells.

Keywords: ER stress; deptor; homeostasis; multiple myeloma; transcription.

Figure 1. Deptor is a nuclear protein

A-B. Immunofluorescence analysis of Deptor expression in plasma cells purified from primary myelomas (A) and in MM cell lines (B). Cells were fixed in 4% formaldehyde for 15 min and then permeabilized with 0.1% Triton X-100. Nuclei were visualized by staining with Hoechst dye. Scale bar represents 10 μm; 63X original magnification. C. Western blot (WB) analysis, with the indicated antibodies (Abs), of total (left), nuclear and cytoplasmic extracts (right) from KMS27, KMS18 and ARH77 MM cell lines. D. WB analysis of soluble nuclear and chromatin-bound protein fractions with the indicated Abs from KMS27 and KMS18 MM cell lines.

Figure 2. Deptor modulates transcription of genes involved in ER homeostasis

A. WB analysis with the indicated Abs of total cell extracts (TCEs) from KMS27 cells transiently transfected with Stealth siRNA negative control (siControl) or siRNA Deptor (siDeptor). B. Volcano Plot of all assembled RNA-seq genes performed with KMS27 cells transiently transfected as in A. The image depicts genes with strong modulation but not significant (qvalue < 0.05) in orange; significant modulation but mild fold-change (abs (log2fc) < 1) in red; significant and strongly modulated genes in green. C. Gene Ontology significance barplot of all siDeptor modulated genes deriving from Cuffdiff analysis. D. Intensity (FPKM) heatmap of endoplasmic reticulum genes (top) and ER stress genes (bottom) modulated after Deptor silencing. E. Correlation analysis of expression values from 550 myeloma patients, as described in Zhan et al [28], between Deptor and endoplasmic reticulum genes (ERLIN2, KEAP1, PSEN2 and DERL3) (probe set 218858_at for DEPTOR). R2 values: Erlin2=0,234; KEAP1=−0,118; PSEN2=−0,318; DERL3=−0,136.

Figure 3. Deptor modulates transcription of genes involved in ER homeostasis

A-B. Quantitative RT–PCR (qRT–PCR) for ER homeostasis gene expression was performed in KMS27 (A) and KMS18 (B) cells transiently transfected with Stealth siRNA negative control (siControl) or siRNA Deptor (siDeptor). Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. *P = 0.002, **P≤0.03 (A); *P≤0.0002, **P≤0.01 (B). C. WB analysis with the indicated Abs of TCEs from KMS27 and KMS18 cells transfected as in A and B. Arrowhead indicates specific ERLIN2 protein band. D. ChIP-qPCR analysis of KMS27 cells using anti-Deptor Ab or control IgGs. Primer were designed to amplified two different promoter regions of CKAP4, ERLIN2 and PSEN2. Error bars represent the standard error of three different experiments. n.s., not significant, *P≤0.0004, **P=0.0225.

Figure 4. Deptor depletion enhances ER stress in MM cells

A. WB analysis with the indicated Abs of TCEs from KMS27 and KMS18 cells treated where indicated with tunicamycin (2mg/ml) or brefeldin A (1μg/ml) for 8hrs. B. WB analysis with the indicated Abs of TCEs from KMS18 and KMS27 cells transiently transfected with Stealth siRNA negative control (siControl) or siRNA Deptor (siDeptor). C. KMS18 and KMS27 cells, transfected as in B, were analyzed by WB with the indicated Abs. D. qRT-PCR for XBP1spl and CHOP mRNA expression was performed after transient transfection of KMS27 cells as in B. Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments performed in duplicate. P≤0.0001. E. KMS27 and KMS18 cells were transfected as in B and then analyzed by WB with the indicated Abs. F. KMS18 and KMS27 cells were transfected as in B and after 60 hrs assayed for cell death by trypan blue staining and percentages represent trypan blue-incorporating cells. Data are presented as the mean SD from three independent experiments performed in duplicate. *P=0.0007, **P=0.0433. G. Left: representative Serum Protein Electrophoresis (SPEP) performed on a Vk*MYC mouse. Asterisks emphasize M-spikes, hallmark of MM disease, detected in mouse serum. Right: WB analysis with the indicated Abs of TCEs of CD138⁺ neoplastic cells from Vk*MYC mice transiently transfected as in B. H. KMS27 cells were transfected as in A and, where indicated, treated with 100nM CCI-779 for 16 hrs and then analyzed by WB using the indicated Abs.

Figure 5. Deptor sensitizes MM cells to Bz treatment

A. Top: WB analysis with the indicated Abs of TCEs from KMS27 cells treated or not with different concentrations of Bz for 4hrs and 8hrs. Bottom: qRT-PCR for Deptor mRNA expression of KMS27 cells treated as in A. Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments performed in duplicate. n.s., not significant, *P<0.03. B. WB analysis with the indicated Abs of TCEs from KMS27 cells transfected with Stealth siRNA negative control (siControl) or siRNA Deptor (siDeptor) and after 24hrs treated or not with 5nM Bz for further 24 and 48hrs. C. Cell death detection of KMS27 cells treated like in B and assayed by trypan blue staining. Percentages represent trypan blue-incorporating cells. Data are presented as the mean SD from three independent experiments performed in duplicate. *P=0.0433, **P≤0.01. D. MM cells overexpressing (KMS27 and KMS18) or lacking (ARH77) of Deptor expression were treated with the indicated concentrations of Bz for 48hrs and cell death was assayed by trypan blue staining. Percentages represent trypan blue-incorporating cells. Data are presented as the mean SD from three independent experiments performed in duplicate. n.s., not significant, *P≤0.01, **P≤0.03.