Targeting Mitochondrial STAT3 with the Novel Phospho-Valproic Acid (MDC-1112) Inhibits Pancreatic Cancer Growth in Mice

<div><p>New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%–97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-x_L, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.</p></div

Proposed mechanism of action of P-V.

<p>Proposed mechanism of action of P-V.</p

Mitochondrial STAT3 overexpression abrogates the anticancer effect of P-V in vivo.

<p>(<b>A</b>) MIA PaCa-2 cells with basal (STAT3<i>^normal</i>) or overexpressed STAT3 (STAT3<i>^high</i>) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 18 days. <i>Top</i>: Images of representative pancreatic tumors. <i>Bottom</i>: Pancreatic tumor weight (mean ± SEM). *<i>p</i><<i>0.01 vs</i>. control; ^#<i>p</i><<i>0.01 vs</i>. STAT3<i>^high</i> P-V-treated group. (<b>B</b>) STAT3 and Mcl-1 expression in STAT3<i>^normal</i> and STAT3<i>^high</i> MIA PaCa-2 orthotopic tumor tissue sections from control and P-V-treated mice (x20). (<b>C</b>) Immunoblots for STAT3 and Bcl-x_L in orthotopic tumor samples. Each lane represents a different tumor sample. Loading control: β-actin. (<b>D</b>) MIA PaCa-2 cells with overexpressed STAT3 Y705F mutant (STAT3<i>^Y705F</i>) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 18 days. Pancreatic tumor weight (mean±SEM). (<b>E</b>) AsPC-1 cells with basal (STAT3<i>^normal</i>) or overexpressed mitochondria-targeted STAT3 (MLS-STAT3) levels were orthotopically implanted in nude mice, which were then treated without (control) or with P-V 150 mg/kg for 21 days. Pancreatic tumor weight (mean±SEM). *<i>p</i><<i>0.01 vs</i>. control; ^#<i>p</i><<i>0.01 vs</i>. MLS-STAT3 P-V-treated group.</p

P-V inhibits STAT3 mitochondrial localization and selectively induces mitochondrial ROS in PC cells.

<p>(<b>A</b>) Immunoblots for STAT3, α-tubulin or COX IV in cytosolic (CF) and mitochondrial (MF) fractions from MIA PaCa-2 cells treated with P-V for 5 h. (<b>B</b>) Immunoblots for STAT3, Hsp90, Hsp60, α-tubulin and COX IV in mitochondrial fractions from MIA PaCa-2 cells treated with P-V, VPA or phospho-aspirin (P-A) for 3 h. (<b>C</b>) Immunoblots for p-STAT3^Ser727, STAT3 and Hsp90 in mitochondrial fractions isolated from MIA PaCa-2 xenografts from control or 150 mg/kg P-V-treated mice. β-tubulin  =  cytosolic cross-contamination control; COX IV  =  mitochondrial loading control. Each lane corresponds to a different tumor sample. (<b>D</b>) P-V decreases the activity of the mitochondrial electron transfer chain complex I (NADPH dehydrogenase). The activity of the mitochondrial complex I was measured as described in Methods. Values are mean±SEM (n = 3); *<i>p</i><<i>0.05 vs</i>. control. (<b>E</b>) MitoSOX Red, DCFDA and DHE fluorescence was measured by flow cytometry in BxPC-3 cells treated with P-V for 1 h. As positive controls, we treated cells with phospho-aspirin (P-A) for 1 h, which induces both DCFDA and DHE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061532#pone.0061532-Zhao1" target="_blank">[13]</a>. <i>Right panel</i>: Mitochondrial O₂⁻ levels in a panel of six PC cells treated with P-V 1.5×IC₅₀ for 1 h. (<b>F</b>) BxPC-3 cells were treated with P-V for up to 2 h, followed by addition of the MitoSOX Red probe and cells were analyzed by confocal microscopy (x40).</p

P-V blocks the mitochondrial membrane potential and induces mitochondrial cell death: The role of mitochondrial STAT3.

<p>(<b>A</b>) Knocking-down STAT3 increases mitochondrial O₂⁻ levels in BxPC-3 cells. Control-siRNA and STAT3-siRNA cells were preloaded with MitoSOX Red, and mitochondrial O₂⁻ was determined by flow cytometry (<i>left</i>) or confocal microscopy (<i>right</i>; x40). (<b>B</b>) STAT3 overexpression reduces the increase in mitochondrial O₂⁻ induced by P-V. BxPC-3 cells were transfected with a STAT3-expressing plasmid or a control plasmid (empty vector) for 48 h, and then treated with P-V for 4 h followed by addition of the probe MitoSOX Red. Cells were then examined by confocal microscopy (x40). (<b>C</b>) P-V collapses the mitochondrial membrane potential (ΔΨm) in a time- (<i>top</i>) and concentration-dependent (<i>bottom</i>) manner. Fluorescence histograms were quantified and results are shown as mean±SEM; *<i>p</i><<i>0.05 vs</i>. control. (<b>D</b>) Immunoblots for cytochrome c, pro-caspases and caspases 9 and 3 in cytosolic protein extracts from BxPC-3 cells treated with P-V. (<b>E</b>) BxPC-3 mitochondria-less (ρ⁰) cells are markedly more resistant to P-V-induced apoptosis. <i>Top</i>: Immunoblots for COX IV and STAT3 in parental (−) and ρ⁰ BxPC-3 cells. Loading control: β-actin.</p

QPCR comparison of the relative abundances of the <i>Bacteroidetes</i> phylum and the <i>Lachnospiriceae</i> clade within the Firmicutes phylum in 6 week old APC^Min/+ and WT mice.

<p>The qPCR assays were conducted using established primers as described in Methods. The median and range of ΔCt values (~Log₂ relative abundance of targeted taxa) are listed for.the ileal, cecal, proximal colonic, distal colonic mucosal samples and the distal colonic luminal samples collected from 30 6 week old APC^Min/+ and 30 wild type mice. The p-values were carried out using the Mann-Whitney U test. The Bonferroni correction was made to the p-value, so that significance was set at p<0.025.</p><p>QPCR comparison of the relative abundances of the <i>Bacteroidetes</i> phylum and the <i>Lachnospiriceae</i> clade within the Firmicutes phylum in 6 week old APC^Min/+ and WT mice.</p

Comparison of phyla and families between wildtype (WT) and APC^Min/+ (APC) mice.

<p>A. The mean relative abundances of phyla (left panel) and families (right panel) as inferred from the 16S rRNA sequence analysis. Only phyla and families with relative abundances >0.5% are shown. The Hotelling T2 test was used to compare the overall microbial composition, with p-values noted above each barchart.</p

Association between gene cluster expression (centroid medians), APC genotype and the relative abundance of Bacteroidetes in the proximal colonic mucosa of 6 week old APC^Min/+ and WT mice.

<p>The cluster medians of seven geneclusters along with APC genotype were used in the following model as described in <i>Methods</i>. The significant effects are <b>bolded</b>, with the threshold set as p-value <0.05. Regression coefficients are also reported as index of effect size.</p><p>Association between gene cluster expression (centroid medians), APC genotype and the relative abundance of Bacteroidetes in the proximal colonic mucosa of 6 week old APC^Min/+ and WT mice.</p

Hierarchical clustering of DEGs in APC^Min/+ proximal colonic RNA transcripts vs. control RNA samples.

<p>One hundred thirty DEGs were selected by edgeR analysis of RNA sequence data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127985#sec002" target="_blank">Methods</a>) and grouped into seven clusters by hierarchical clustering. Shown on the left are the three upregulated clusters (1–3), and shown on the right are the four dowregulated clusters (4–7).</p><p>Hierarchical clustering of DEGs in APC^Min/+ proximal colonic RNA transcripts vs. control RNA samples.</p