Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models

Depletion of the Aryl Hydrocarbon Receptor in MDA-MB-231 Human Breast Cancer Cells Altered the Expression of Genes in Key Regulatory Pathways of Cancer

<div><p>The aryl hydrocarbon receptor (AhR), a transcription factor that is best known for its role in mediating the toxic responses elicited by poly aromatic hydrocarbons as well as many other environmental factors; is also involved in breast cancer progression. We previously reported that stable knockdown of AhR decreased the tumorigenic properties of the highly metastatic MDA-MB-231 breast cancer cell line; whereas ectopic overexpression of AhR was sufficient to transform immortalized human mammary epithelial cells to exhibit malignant phenotypes. In the present study we investigated the genes that are differentially regulated by AhR and are controlling cellular processes linked to breast cancer. We used Affymetrix Human GeneChip 1.0-ST whole transcriptome arrays to analyze alterations of gene expression resulting from stable AhR knockdown in the MDA-MB-231 breast cancer cell line. The expression of 144 genes was significantly altered with a ≥2.0-fold change and a multiple test corrected p-value ≤0.05, as a result of AhR knockdown. We demonstrate that AhR knockdown alters the expression of several genes known to be linked to cancer. These genes include those involved in tryptophan metabolism <i>(KYNU)</i>, cell growth (<i>MUC1</i> and <i>IL8),</i> cell survival (<i>BIRC3</i> and <i>BCL3</i>), cell migration and invasion (<i>S100A4</i> and <i>ABI3),</i> multi-drug resistance (<i>ABCC3</i>) and angiogenesis (<i>VEGFA</i> and <i>CCL2</i>). The identification of the genes and pathways affected by AhR depletion provides new insight into possible molecular events that could explain the reported phenotypic changes. In conclusion AhR knockdown alters the expression of genes known to enhance or inhibit cancer progression; tipping the balance towards a state that counteracts tumor progression.</p></div

List of genes related to phenotypic changes observed following AhR knockdown in MDA-MB-231 cells.

1<p>P-value <0.05 and fold change ≥2.0 was considered significant.</p>2<p>The fold changes in transcriptome levels and significance were calculated using one-way ANOVA.</p><p>KEGG pathway analysis was performed using the WebGESTALT online tool. List identified genes which had significant alterations following AhR knockdown¹.</p

Subnetwork of functional interactions of AhR-target networks.

<p>Initial nodes were populated with differential expressed genes. Circles represent genes of AhR targets, while diamonds represent linker genes.</p

Validation of microarray data by qRT-PCR.

<p>Differential gene expression from the microarrays analyses was confirmed by qRT-PCR of 10 selected genes using gene specific primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100103#pone-0100103-t001" target="_blank">Table 1</a>. Data represents mean of triplicates, normalized to GAPDH, and presented as relative fold-change (RFC) of Clone 8 to that of Scr-control cells.</p

Hierarchical clustering of differentially expressed genes in MDA-MB-231 cells following AhR knockdown.

<p>(A) Illustration of the procedure used to examine differential gene expression following AhR knockdown. (B) Clusters of genes altered by AhR KD. Heat map reveals clusters of genes. Green indicates genes up-regulated compared to control cells and red indicates genes down-regulated compared to control cells.</p

Functional enrichment of KEGG pathway analysis following AhR knockdown in MDA-MB-231 cells.

1<p>Ratio of enrichment refers to the number of observed genes divided by the number of expected genes from each KEGG category.</p>2<p>Multiple test adjustment (BH) FDR adjusted p<0.05 was considered significant.</p><p>KEGG pathway analysis was performed using the WebGESTALT online tool. It identified those pathways that had significant alterations following AhR knockdown.</p

Functional enrichment of GO biological processes following AhR knockdown in MDA-MB-231 cells (C8).

<p>*Counts refer to the number of genes from the input list that fit into the given pathway.</p><p>Enrichment was performed using the WebGESTALT online tool. It identified those biological processes that had significant alterations following AhR knockdown.</p

List of primers used for validation of microarray data by quantitative reverse transcriptase-polymerase chain reaction.

<p>F, forward; R, reverse.</p

Top 10 genes significantly up- or down-regulated in MDA-MB-231 cells following AhR KD.

<p>Top 10 genes significantly up- or down-regulated in MDA-MB-231 cells following AhR KD.</p