Prognostic value and targeted inhibition of survivin expression in esophageal adenocarcinoma and cancer-adjacent squamous epithelium

Background: Survivin is an inhibitor of apoptosis and its over expression is associated with poor prognosis in several malignancies. While several studies have analyzed survivin expression in esophageal squamous cell carcinoma, few have focused on esophageal adenocarcinoma (EAC) and/or cancer-adjacent squamous epithelium (CASE). The purpose of this study was 1) to determine the degree of survivin up regulation in samples of EAC and CASE, 2) to evaluate if survivin expression in EAC and CASE correlates with recurrence and/or death, and 3) to examine the effect of survivin inhibition on apoptosis in EAC cells. Methods: Fresh frozen samples of EAC and CASE from the same patient were used for qRT-PCR and Western blot analysis, and formalin-fixed, paraffin-embedded tissue was used for immunohistochemistry. EAC cell lines, OE19 and OE33, were transfected with small interfering RNAs (siRNAs) to knockdown survivin expression. This was confirmed by qRT-PCR for survivin expression and Western blot analysis of cleaved PARP, cleaved caspase 3 and survivin. Survivin expression data was correlated with clinical outcome. Results: Survivin expression was significantly higher in EAC tumor samples compared to the CASE from the same patient. Patients with high expression of survivin in EAC tumor had an increased risk of death. Survivin expression was also noted in CASE and correlated with increased risk of distant recurrence. Cell line evaluation demonstrated that inhibition of survivin resulted in an increase in apoptosis. Conclusion: Higher expression of survivin in tumor tissue was associated with increased risk of death; while survivin expression in CASE was a superior predictor of recurrence. Inhibition of survivin in EAC cell lines further showed increased apoptosis, supporting the potential benefits of therapeutic strategies targeted to this marker. © 2013 Malhotra et al

Chemotherapy by Survivin Expression Level.

<p>*2 patients received concurrent chemoradiation.</p

Cox Regression Survival Analysis.

1<p>In pack year.</p>2<p>At time of esophagectomy.</p>3<p>History of Barrett's Esophagus.</p>4<p>High risk group determined by adjacent squamous epithelial tissue mean Survivin levels.</p>5<p>High risk group determined by tumor tissue mean Survivin levels.</p

Stage of Tumor by Survivin Expression Level.

<p>Stage of Tumor by Survivin Expression Level.</p

ROC analysis to correlate survivin levels and recurrence.

<p>The probability for distant recurrence was determined for survivin levels in human EAC tumor and adjacent squamous epithelial tissue. An increased survivin level in adjacent squamous epithelial tissue was determined to be a risk factor for distant recurrence (p = 0.02).</p

A: Reverse transcriptase polymerase chain reaction of siRNA inhibition of survivin in EAC cell line.

<p>OE19 and OE33 EAC cell lines were transfected with siRNA and survivin expression was analyzed between control and transfected cell lines. Both cell lines transfected with siRNA showed clear inhibition and downregulation of survivin expression. <b>B: Western blot of siRNA inhibition of survivin in EAC cell line.</b> siRNA was incubated with OE19 and OE33 cell lines to inhibit survivin expression. β-Actin was used as a loading control. Survivin was downregulated, while downstream apoptotic proteins cleaved caspase 3 and cleaved PARP were upregulated. The upregulation of downstream apoptotic proteins indicates apoptosis occurs through incubation with siRNA.</p

Characteristics of the 37 patients included in the study.

<p>Characteristics of the 37 patients included in the study.</p

Survivin inhibition pathway.

<p>Survivin binds to and inhibits caspase 9, caspase 9 is unable to cleave caspase 3, caspase 3 is unable to cleave PARP, PARP promotes DNA repair and does not induce apoptosis.</p

Quantitative real time polymerase chain reaction (qRT-PCR) of patient survivin levels.

<p>A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.</p

Immunohistochemistry of survivin in human tissue.

<p>Survivin expression was evaluated through IHC staining, tumor tissue and adjacent squamous epithelium showed presence of staining indicative of survivin expression. Arrows represent positive staining.</p