Erythropoietin Stimulates Tumor Growth via EphB4

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo’s effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin

Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer

Down-Regulation of 5-HT_1B and 5-HT_1D Receptors Inhibits Proliferation, Clonogenicity and Invasion of Human Pancreatic Cancer Cells

<div><p>Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT_1B and 5-HT_1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT_1B and 5-HT_1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT_1B and 5-HT_1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT_1B and 5-HT_1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT_1B– and 5-HT_1D–mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT_1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.</p></div

Effect of down-regulation of 5-HT_1B and 5-HT_1D receptors expression on the invasion/migration of PaCa cells.

<p>PANC-1 cells (<b>A</b>) and MIAPaCa-2 cells (<b>B</b>) were transfected with 50 nM of indicated siRNAs (for 72 h), and equal numbers of viable cells were seeded onto Matrigel-coated Transwell filters in Matrigel invasion chambers. The number of the cells that invaded after 24 h was determined as in protocol. Magnification,100×. The histograms show the mean of percentages of invasion ± SD of three experiments. * P<0.05 vs. control cells. (<b>C</b>) The involvement of 5-HT_1B and 5-HT_1D receptors in regulation of PANC-1 cell motility as analyzed by the wound healing assay. A single scratch was made in the center of the confluent cell monolayer, and the wounded monolayers were transfected with indicated siRNAs. The wounds repair was monitored for 24 h and visualized microscopically with original magnification ×100. Images were taken immediately (0 h), and after 12 h and 24 h of scratching the cultures. The histogram shows the percentages of the cells migration, and the data is expressed as mean of the percentages of migration ± SD of three independent experiments. * represents significant difference between indicated groups (P<0.05).</p

Effects of down-regulation of 5-HT_1B and 5-HT_1D receptors on PaCa cell proliferation.

<p>(<b>A</b>) 5-HT_1B and 5-HT_1D receptors are highly expressed in several pancreatic cancer cell lines. Cell lysates of several PaCa cell lines and normal human pancreatic duct epithelial (HPDE) cells were subjected to Western blot analysis as indicated. β-actin was used as a loading control. (<b>B–C</b>) 5-HT_1B and 5-HT_1D receptors expression levels in PANC-1 (B) and MIAPaCa-2 cells (C) after knockdown of the receptors expression with their corresponding siRNAs. Cells were transfected with 50 nM of indicated siRNAs, and 72 h later, cell lysates were subjected to Western blot analysis. β-actin was used as loading control. The histograms show the relative quantification of the indicated proteins levels. (<b>D–E</b>) mRNA expression of 5-HT_1B and 5-HT_1D receptors in PANC-1 (D) and MIAPaCa-2 cells (E) after knockdown of the receptors expression with their corresponding siRNAs. Cells were transfected with 50 nM of indicated siRNAs, and 72 h later, the total RNA was extracted and the transcript levels of 5-HT_1B and 5-HT_1D were determined by standard RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105245#s2" target="_blank">Materials and Methods</a>. GAPDH was used as loading control. (<b>F–G</b>) siRNA-mediated 5-HT_1B and 5-HT_1D receptors knockdown inhibits PaCa cells proliferation. PANC-1 (F) and MIAPaCa-2 cells (G) were transfected with 50 nM of indicated siRNAs, and after 72 h, proliferation was detected by an MTS assay. Data are represented as mean ± SD. * P<0.05 vs. control cells. All experiments were independently performed three times.</p

5-HT_1B/1D receptors-induced regulation of TG2/NF-κB signaling might mediate proliferation/invasion-promoting pathways.

<p>(<b>A</b>) 5-HT_1B and 5-HT_1D receptors regulate TG2 and NF-κB expression. PANC-1 cells were treated with indicated siRNAs as described above. (<b>B</b>) siRNA-mediated TG2 knockdown induced downstream molecular effects similar to that observed after knockdown of 5-HT_1B and 5-HT_1D receptors expression. Cells were transfected with 50 nM of TG2 siRNA or control siRNA, and cell lysates were subjected to Western blot analysis. β-actin was used as loading control. (<b>C</b>) Inhibition of constitutive activation of NF-κB inhibits the key signaling promoting proliferation/invasion and decreases the mesenchymal markers, Fibronectin and α-SMA. Cells were treated with indicated concentration of NF-κB activation Inhibitor II, JSH-23, for 24 h, and cell lysates were subjected to Western blot analysis. Tubulin was used as loading control. Three independent experiments were performed with similar results, and representative data is shown.</p

The postulated molecular regulation of 5-HT_1B and 5-HT_1D receptors to β1 integrin/Src/FAK complex, ECM/uPAR/MMP-2 signaling and the zinc finger transcriptional regulators of EMT in PaCa cells.

<p>These receptors-regulated signaling might be mediated through TG2/NF-κB axis. 5-HT_1B and 5-HT_1D receptors mediate β1-integrin activity to recruit a Src–FAK complex promoting the cell proliferation and migration. Upon different extracellular mitogenic stimuli (e.g; growth factors, hormones and neurotransmitters), the over-expressed 5-HT_1B/1D receptors promote the activation of urokinase plasminogen activator receptor (uPAR), and matrix metalloproteinase (MMP-2), facilitating extra-cellular matrix degradation and enhancement of invasion process. Also, 5-HT_1B/1D receptors stimulate the expression of zinc finger transcriptional factors (Snail and TCF8/ZEB1). These mesenchyme markers, in turn, repress the gene transcription of epithelial markers (claudin-1 and E-cadherin) leading to stimulation of epithelial mesenchymal transition (EMT), further supporting the proliferation and invasiveness of PaCa cells.</p

Effect of targeting 5-HT_1B and 5-HT_1D receptors on PaCa cell clonogenicity.

<p>PANC-1 (<b>A</b>) and MIAPaCa-2 cells (<b>B</b>) were transfected (once/week) with indicated siRNAs. The cells were incubated for 14 days, the colonies were stained with crystal violet and the colonies-area distribution regions were measured densitometrically at the end of the 14 days. The histograms show the percentages of the formed colonies, after 14 days of the first transfection. Data is expressed as mean of percentages of colony formation ± SD of three independent experiments. * P<0.05 vs. control cells.</p

Downstream molecular effects of knockdown of the expression of 5-HT_1B and 5-HT_1D receptors, on invasion and proliferation biomarkers in PaCa cells.

<p>(<b>A–B</b>) The effect of siRNAs-mediated 5-HT_1B and 5-HT_1D receptors down-regulation on β1 integrin/ECM-mediated downstream signaling in PANC-1 (A) and MIAPaCa-2 cells (B). (Upper panel) Cells were transfected with 50 nM of indicated siRNAs, and after 72 h, the total RNA was extracted and the transcript levels of 5-HT_1B and 5-HT_1D were determined by standard RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105245#s2" target="_blank">Materials and Methods</a>. GAPDH was used as loading control. (Lower panel) Cells were transfected with 50 nM of indicated siRNAs, and after 72 h, cell lysates were subjected to Western blot analysis. β-actin was used as loading control. (<b>C–D</b>) The effect of 5-HT_1B and 5-HT_1D receptors down-regulation on EMT process in PANC-1 (C) and MIAPaCa-2 cells (D). Silencing of 5-HT_1B and 5-HT_1D receptors significantly increase the expression of EMT tight junction protein, claudin-1, and decrease the zinc finger ZEB1 transcriptional factors, TCF8 and Snail. The cells treated as in (A–B). Three independent experiments were performed with similar results, and representative data is shown. (<b>E</b>) Heat map clustered from RPPA analysis, and a histogram of the normalized fold changes of the selected EMT-markers in PANC-1 cells treated with 50 nM of indicated siRNAs for 72 h. Knockdown of 5-HT_1B and 5-HT_1D receptors increase the epithelial marker, E-cadherin, and decrease its transcriptional repressor (TWIST).</p