INHBA Overexpression Promotes Cell Proliferation and May Be Epigenetically Regulated in Esophageal Adenocarcinoma Christopher W. Seder, MD, Wibisono.

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INHBA Overexpression Promotes Cell Proliferation and May Be Epigenetically Regulated in Esophageal Adenocarcinoma Christopher W. Seder, MD, Wibisono Hartojo, MD, Lin Lin, MD, PhD, Amy L. Silvers, PhD, Zhuwen Wang, MS, MD, Dafydd G. Thomas, MD, PhD, Thomas J. Giordano, MD, PhD, Guoan Chen, MD, PhD, Andrew C. Chang, MD, Mark B. Orringer, MD, David G. Beer, PhD Journal of Thoracic Oncology Volume 4, Issue 4, Pages 455-462 (April 2009) DOI: 10.1097/JTO.0b013e31819c791a Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Oligonucleotide microarray analysis of 46 esophageal specimens demonstrated at least two-fold expression in 11 of 15 (73.3%) and four-fold expression in 9 of 15 (60%) of esophageal adenocarcinoma (EAC) relative to Barrett's metaplasia (BM). x axis represents tumor identifier; y axis represents transcript expression relative to BM. BM, Barrett's metaplasia; LGD, low-grade dyslasia; HGD, high-grade dyslasia; EAC, esophageal adenocarcinoma. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of 45 esophageal specimens used in the oligonucleotide microarray (Figure 1) confirming overexpression of INHBA in esophageal adenocarcinoma (EAC) relative to Barrett's metaplasia (BM). x axis represents tumor identifier; y axis represents transcript expression relative to BM. BM, Barrett's metaplasia; LGD, low-grade dyslasia; HGD, high-grade dyslasia; EAC, esophageal adenocarcinoma. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Representative sections of tissue microarray. A, and (B) Inhibin βA protein staining was not detected in most Barrett's metaplasia, but was seen in (C) and (D) 69.6% of esophageal adenocarcinomas by immunohistochemistry. A, and (D) ×40 magnification; B, ×20 magnification; C, ×10 magnification. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 A, FLO and (B) OE-33 cells treated with activin demonstrated increased proliferation at 96 hours as assessed using WST-1 assays. The effect of activin treatment was greater in calf serum (CS) than in fetal bovine serum (FBS). All experiments repeated in quadruplicate. *p < 0.05. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 OE-33 cells treated with (A) activin inhibitor follistatin and (B) 10 nM INHBA-targeting siRNA demonstrated reduced proliferation at 96 hours relative to mock controls using WST-1 assays. Repeat transfection after 48 hours resulted in further growth-inhibition relative to controls. All experiments repeated in quadruplicate. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 6 INHBA mRNA production in (A) FLO was up-regulated upon treatment with histone deacetylase inhibitor trichostatin A (TSA) and demethylating agent 5-aza-2′deoxycytidine (5-AZA). B, A significantly blunted response was seen upon exposure of OE-33 to TSA and 5-AZA. Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 7 Immunohistochemistry of A, untreated FLO cells did not demonstrate inhibin βA protein expression. B, Treatment of FLO cells for 12 hours with trichostatin A (TSA) significantly up-regulated inhibin βA protein expression (×40 magnification). Journal of Thoracic Oncology 2009 4, 455-462DOI: (10.1097/JTO.0b013e31819c791a) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions