Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib

Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.Peer reviewe

MiR-200c Regulates Noxa Expression and Sensitivity to Proteasomal Inhibitors

The pro-apoptotic p53 target Noxa is a BH3-only protein that antagonizes the function of selected anti-apoptotic Bcl-2 family members. While much is known regarding the transcriptional regulation of Noxa, its posttranscriptional regulation remains relatively unstudied. In this study, we therefore investigated whether Noxa is regulated by microRNAs. Using a screen combining luciferase reporters, bioinformatic target prediction analysis and microRNA expression profiling, we identified miR-200c as a negative regulator of Noxa expression. MiR-200c was shown to repress basal expression of Noxa, as well as Noxa expression induced by various stimuli, including proteasomal inhibition. Luciferase reporter experiments furthermore defined one miR-200c target site in the Noxa 3′UTR that is essential for this direct regulation. In spite of the miR-200c:Noxa interaction, miR-200c overexpression led to increased sensitivity to the clinically used proteasomal inhibitor bortezomib in several cell lines. This apparently contradictory finding was reconciled by the fact that in cells devoid of Noxa expression, miR-200c overexpression had an even more pronounced positive effect on apoptosis induced by proteasomal inhibition. Together, our data define miR-200c as a potentiator of bortezomib-induced cell death. At the same time, we show that miR-200c is a novel negative regulator of the pro-apoptotic Bcl-2 family member Noxa

MiR-200c potentiates proteasome inhibitor-mediated cell death.

<p>(<b>A</b>) HCT116 cells were transfected with pre-miR-200c or pre-miR-control oligos for 24 hours, treated with indicated concentrations of bortezomib for an additional 24 hours and processed for immunoblotting for GAPDH, Noxa, cleaved PARP and cleaved caspase 3. Protein size in kilodaltons (kDa) is also shown. (<b>B</b>) HCT116 cells were transfected with pre-miR-200c or pre-miR-control oligos for 48 hours and apoptosis was assessed by Annexin V/PI staining and FACS analysis. (<b>C</b>) HCT116 cells were treated with 20 nM bortezomib as in (A) and apoptosis was assessed by Annexin V/PI staining and FACS analysis. Graphs show the mean of percentages of Annexin V-positive cells, including PI-positive and PI-negative, from three independent experiments.</p

MiR-200c represses Noxa protein under both normal conditions and during cellular stress.

<p>(<b>A</b>) HEK293 cells were transfected with a plasmid encoding the miR-200c microRNA cluster or an empty vector control. Cells were collected at indicated timepoints, whole cell extracts were prepared and subjected to immunblotting for Noxa. (<b>B</b>) The indicated cell lines were transfected with pre-miR-200c or pre-miR-control oligos for 48 hours, whole cell extracts were prepared and analyzed for Noxa protein expression. (<b>C</b>) HEK293 cells were transfected with the indicated expression constructs for 24 and 48 hours respectively, and Noxa mRNA expression was determined by qRT-PCR analysis. Noxa mRNA expression was normalized to that of GAPDH. (<b>D</b>) HEK293 cells were transfected with indicated expression constructs (<i>left panel</i>) or oligonucleotides (<i>right panel</i>). 24 hours post-transfection, cells were treated with the indicated concentration of MG132 for an additional 3 or 24 hours, and processed for Noxa immunoblotting as in (A). GAPDH was used as a loading control in all immunoblotting experiments. Protein size in kilodaltons (kDa) is also shown.</p

Noxa attenuates the proapoptotic effect of miR-200c.

<p>(<b>A</b>) HCT116 cells were transfected with pre-miR-200c or pre-miR-control oligos together with Noxa or GFP control siRNA oligos for 24 hours. Cells were treated with 20 nM bortezomib for an additional 24 hours and apoptosis was assessed by Annexin V/PI staining and FACS (<i>upper panel</i>). Cells were also collected and processed for immunoblotting for GAPDH and Noxa to demonstrate efficiency of siRNA knockdown (<i>lower panel</i>). Protein size in kilodaltons (kDa) is shown. (<b>B</b>) HCT116 cells were transfected with the indicated constructs and oligos. Samples were either left untreated (<i>left panel</i>) or treated with 20 nM bortezomib (<i>right panel</i>) and were subsequently analyzed as in (A). Graphs show the mean of percentages of Annexin V-positive cells, including PI-positive and PI-negative, from three independent experiments. Percentage values above graphs show fold increase in apoptosis when comparing indicated samples.</p

MiR-200c directly targets the Noxa 3′UTR.

<p>(<b>A</b>) The extent of evolutionary conservation of the predicted miR-200c target site in the Noxa 3′UTR is shown. Outlined box demarks region complimentary to the seed sequence of miR-200c. (<b>B</b>) Specific mutations introduced at the miR-200c target position in the Noxa 3′UTR reporter. (<b>C</b>) The indicated constructs were introduced into HEK293 cells and luciferase activity was measured. The luciferase ratio between the Renilla and Firefly of the empty vector transfection was adjusted to 1. (<b>D</b>) Luciferase reporter experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036490#pone-0036490-g001" target="_blank">Figure 1A</a> with the wild-type Noxa 3′UTR or the seed sequence mutant reporter constructs. The luciferase ratio between the Renilla and Firefly of the wild type Noxa 3′UTR vector transfection was adjusted to 1.</p

MiR-200c is a candidate Noxa-regulating microRNA.

<p>(<b>A</b>) The Noxa 3′UTR is repressed in MCF7 cells. The pMIR-REPORT (<i>upper panel</i>) and psiCHECK2 (<i>lower panel</i>) vectors with the full length Noxa 3′UTR downstream of luciferase or empty vector controls were introduced into the indicated cell lines. Luciferase activity was normalized to the activity of an external Renilla luciferase plasmid (<i>upper panel</i>) or to an internal Firefly luciferase (<i>lower panel</i>). (<b>B</b>) Expression profiling of microRNAs predicted to target the Noxa 3′UTR. (<b>C</b>) The repressive element is located in the distal part of the Noxa 3′UTR. The full length Noxa 3′UTR luciferase plasmid or the indicated deletion mutants were introduced into MCF7 cells and luciferase activity was measured as in (A). A schematic representation of the different 3′UTR constructs used in this study is also shown. The location of target sites of the three Noxa-regulating candidates is included in the scheme. (<b>D</b>) Expression of Noxa inversely correlates with that of miR-200c. MiR-200c expression was determined by qRT-PCR analysis in the indicated cell lines. Expression was normalized to that of the small nucleolar RNA RNU48 (<i>upper panel</i>). Protein extracts were prepared in parallel and subjected to immunoblotting for endogenous Noxa (<i>lower panel)</i>. GAPDH was used as a loading control. Protein size in kilodaltons (kDa) is also shown.</p