Abstract LB-152: Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis.
Authors: Carruba Giuseppe , Miceli Vitale , Cocciadiferro Letizia , Polito Lucia M. , Agostara Biagio , Granata Orazia M. 
Published: August 2014

Abstract
Although there is evidence that hints at a potential role of estrogen in hepatocellular carcinoma (HCC), the use of antiestrogens has failed to improve survival of patients. We have investigated aromatase-driven estrogen formation in nontumoral and malignant human liver tissues and cells, also in relation to the expression of estrogen receptor (ER) α, ERβ and their splicing variants and of amphiregulin (AREG), an EGFR ligand, aiming to get insights in the potential role of estrogen and the underlying mechanism(s) in HCC. Chromatographic and exon-specific RT-PCR analyses were used to assess activity and expression of aromatase and the expression of wild-type (hERα66, hERβ1) and variant (hERα46, hERα36; hERβ2/Cx, hERβ5) ER, both in vivo and in vitro. Following incubation of tissue minces or cells with either testosterone or androstenedione used as precursor, human HCC tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates of 20% at 24h and &gt;95% at 72h. By contrast, no aromatase activity could be detected in nontumoral hepatic tissues and HA22T liver cancer. Cirrhotic samples exhibited variable enzyme activity, with average estrogen formation rates of 4-8%, while Huh7 HCC cells gave rise to 34% estrogen formation. Markedly lower aromatase mRNA levels were observed in HA22T cells and nontumoral liver tissues, as compared to HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels. Interestingly, no or low expression of wild type ERα and ERβ could be detected in liver cancer cells and malignant tissues. Conversely, equivalent amounts of the hERα46 variant were observed in both cells and tissues, while hERα36 expression was highest in HepG2 cells and HCC samples, intermediate in Huh7 cells and cirrhotic tissues, and very low in HA22T cells and nontumoral liver. Only occasional expression of the two ERβ variants was observed in liver cancer cells and tissues. It is noteworthy that the pattern of hERα36 expression was strictly related to that of aromatase, suggesting that this ERα variant may be primarily implicated as a mediator of estrogen action in the malignant liver. AREG expression was consistently associated with that of aromatase, with nearly 3-fold and 8-fold higher levels being seen in HepG2 cells than in Huh7 cells (P=0,002) and HA22T cells (P=0,0014), respectively. This evidence suggests that AREG expression may be upregulated by estrogens in human HCC by binding to the membrane-associated ERα36 variant. Based on present and previous experimental evidence, we propose here a unifying hypothetical model whereby locally elevated, aromatase-driven, estrogen formation, combined with a shift in ER wild type/variant expression and the activation of rapid AREG signaling, may be primarily implicated in development and/or progression of human HCC and other neoplastic or chronic human diseases.
Citation Format: Giuseppe Carruba, Vitale Miceli, Letizia Cocciadiferro, Lucia M. Polito, Biagio Agostara, Orazia M. Granata. Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2013-LB-152