Cohen Greg, Mustafi Reba, Chumsangsri Anusara, Little Nathaniel, Nathanson Jeff, Cerda Sonia, Jagadeeswaran Sujatha, Dougherty Urszula, Joseph Loren, Hart John, Yerian Lisa, Tretiakova Maria, Yuan Weihua, Obara Piotr, Khare Sharad, Sinicrope Frank A, Fichera Alessandro, Boss Gerry R, Carroll Robert, Bissonnette Marc
Department of Medicine, University of Chicago, Illinois, USA.
Cancer Res. 2006 Jun 1;66(11):5656-64. doi: 10.1158/0008-5472.CAN-05-0308.
Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA >/=2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 +/- 1.7-fold), transforming growth factor-alpha (14.4 +/- 5.0-fold), heparin-binding EGF-like growth factor (4.5 +/- 1.4-fold), cyclin D1 (4.6 +/- 0.7-fold), and cyclooxygenase-2 (COX-2; 9.3 +/- 4.2-fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 +/- 1.2-fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 +/- 1.3-fold), phosphoactive ErbB2 (2.6 +/- 0.4-fold), phosphoactive extracellular signal-regulated kinase (3.7 +/- 1.1-fold), and cyclin D1 (3.4 +/- 0.8-fold; P < 0.05). Ras was activated in 46% of ACF (3.2 +/- 0.4-fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF.
异常隐窝灶（ACF）是具有异质性分子和病理特征的异常结肠隐窝集合。大的发育异常ACF是结肠癌的假定前体，其肿瘤风险与增殖增加有关。在本研究中，我们研究了表皮生长因子受体（EGFR）信号传导在调节ACF增殖中的作用。使用放大染色内镜检查，我们收集了具有发育异常内镜特征的大ACF，并分别对相邻黏膜进行活检。通过实时PCR测量转录水平，通过蛋白质印迹评估蛋白质，并将水平表示为相邻黏膜的倍数变化。通过PCR评估K-ras和B-Raf突变，并通过Ras-GTP /（Ras-GTP + Ras-GDP）的比率评估Ras激活。在RNA水平上，38%的ACF增殖过度，增殖细胞核抗原（PCNA）mRNA是相邻黏膜的2倍或更高。增殖过度的ACF中，EGFR（6.0±1.7倍）、转化生长因子-α（14.4±5.0倍）、肝素结合表皮生长因子样生长因子（4.5±1.4倍）、细胞周期蛋白D1（4.6±0.7倍）和环氧合酶-2（COX-2；9.3±4.2倍；P<0.05）的mRNA水平显著升高。在蛋白质水平上，46%的ACF增殖过度（PCNA，3.2±1.2倍）。在增殖过度的ACF中，44%的四种EGFR信号传导成分显著增加：EGFR（9.5±1.3倍）、磷酸化活性ErbB2（2.6±0.4倍）、磷酸化活性细胞外信号调节激酶（3.7±1.1倍）和细胞周期蛋白D1（3.4±0.8倍；P<0.05）。46%的ACF中Ras被激活（3.2±0.4倍；P<0.05），但只有7%的ACF存在K-ras突变。与COX-2 mRNA相反，增殖过度的ACF中该蛋白并未增加。总之，我们已经表明，PCNA上调的ACF具有增加的EGFR信号传导成分，这可能导致外观发育异常的ACF增殖状态增强。
