Conversations under a Tung Tree

<p>Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of <i>SFRP3</i> in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate <i>SFRP3</i> expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, <i>SFRP3</i> mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of <i>SFRP3</i> in LUAD and LUSC patients. Moreover, DNA hypermethylation of <i>SFRP3</i> was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of <i>SFRP3</i> expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and <i>in vitro</i> demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of <i>CyclinD1</i> expression <i>in vitro</i>. Our results indicate that <i>SFRP3</i> acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.</p

Correlation analysis of mRNA expression in human breast cancer tissues.

<p>(A) By comparing the <i>BDNF</i> and <i>SFRP1</i> mRNA expression of 87 tumor samples a significant correlation was found (Spearman coefficient, p<0.05). (B) A correlation between LY96 and SFRP1 mRNA expression in 86 human breast cancer tissues could not be detected. n.s.: not significant.</p

The clinical impact of <i>SFRP1/BDNF</i> gene expression in human breast cancer.

<p>Using the KMPLOT data set an association between unfavorable clinical outcome for breast cancer patients and (<b>A</b>) <i>SFRP1</i> expression, as well as (<b>B</b>) <i>BDNF</i> expression and (<b>C</b>) a combination of abundant <i>SFRP1/BDNF</i> expression was observed.</p

Selected GO categories of the microarray analysis independent of the different breast cancer subtypes.

<p>Selected GO categories of the microarray analysis independent of the different breast cancer subtypes.</p

Selected GO categories of the microarray analysis of the luminal-like SKBR3 tumor model.

<p>Selected GO categories of the microarray analysis of the luminal-like SKBR3 tumor model.</p

Multivariate Cox regression analysis including all factors potentially influencing RFS.

a<p>Median <i>BDNF</i> mRNA expression = 4.25 (low: ≤ median expression, high: >median expression);</p>b<p>According to clinical data of the TCGA data set <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102558#pone.0102558-The1" target="_blank">[17]</a>; Significant P-values are marked in bold face.</p

Analysis of SFRP1 and BDNF expression in human breast tissues.

<p>(A) Immunohistochemical staining of human breast tissues is shown exemplarily. SFRP1 and BDNF protein expression of tissue samples from the same patient (a and d, b and e) is shown. c and f: negative controls of SFRP1 and BDNF immunohistochemical staining, respectively. Scale bar: 100 µm. (B) In the left columns three tissues (patient #1 - #3) exhibit no or weak staining of SFRP1 and BDNF whereas the right columns represent three tissues (patient #4 - #6) that have a strong SFRP1 and BDNF protein expression. These images illustrate exemplarily the correlation of SFRP1 and BDNF protein expression <i>in vivo</i>. Scale bars: 100 µm.</p

BDNF re-expression mediates reduced cell proliferation in BT20 breast cancer cells.

<p>(A) Stable cell clones with a full-length cDNA of <i>BDNF</i> show abundant re-expression of <i>BDNF</i> mRNA while empty pT-Rex-DEST30 vector controls completely lack <i>BDNF</i> mRNA. (B) In concordance, mock clones are negative for BDNF protein whereas the 25 kDa BDNF protein is strongly expressed in stable BDNF clones. (C) XTT assay was performed at four subsequent time points. The baseline level at 24 h for each clone was set to 1. A slight decrease in cell proliferation (58.4%) was observed in the stable BDNF clones. (D) Proliferation is significantly (p<0.001) reduced in BT20 breast cancer cells re-expressing BDNF.</p

Generation of human breast cancer cell lines stably expressing SFRP1.

<p>Stable clones have been generated with a full-length SFRP1 cDNA or with empty pEF6/V5 vector control. (A) Semi-quantitative real-time PCR for SFRP1 re-expression was performed after transfection in BT20 and (B) SKBR3 cells. <i>SFRP1</i> mRNA was only detectable in the SFRP1 clones. (C) Western blot analysis was performed on lysates of three BT20 and (D) SKBR3 mock and of three SFRP1 clones. SFRP1 protein expression increased remarkably after transfection with a SFRP1 expression vector compared to the corresponding mock vector. β-actin was used as a loading control.</p

Selected GO categories of the microarray analysis of the basal-like BT20 tumor model.

<p>Selected GO categories of the microarray analysis of the basal-like BT20 tumor model.</p