Abstract
Hepatocarcinogenesis is a multistep process driving the progressive transformation of normal liver cells into highly malignant derivatives. Unlimited proliferation and telomere maintenance have been recognized as prerequisites for the development of liver cancer. Moreover, recent studies identified illegitimate β-catenin signaling as relevant hit in a considerable subset of patients. To further investigate the currently not well-understood malignant evolution driven by telomerase and β-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of β-catenin signaling. As expected, constitutive activation of β-catenin signaling significantly enhanced proliferation with decreasing serum dependence. Previously intact contact inhibition was almost completely eliminated. Interestingly, after several passages in cell culture, immortalized clones with dominant-positive β-catenin signaling acquired additional chromosomal aberrations, in particular translocations, anchorage-independent growth capabilities, and formed tumors in athymic nude mice. In further support for the driving role of β-catenin during hepatocarcinogenesis, improved colony growth in soft agar and accelerated tumor formation was also confirmed in Huh7 cells following stable expression of the constitutively active S33Y β-catenin mutant. Telomerase inhibition showed that short-term expansion of transformed clones was not telomerase dependent. Finally, cancer pathway profiling in derived tumors revealed upregulation of characteristic genes associated with invasion and angiogenesis. In conclusion, illegitimate activation of β-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for β-catenin signaling in the initial phase of hepatocarcinogenesis.
Highlights
Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide with continuously rising incidence rates in Western countries [1, 2]
Transfection of FH-human telomerase reverse transcriptase (hTERT) with the constitutively active S33Y b-catenin mutant as well as with the wild-type control resulted in a significant increase in b-catenin transcripts determined by quantitative PCR (qPCR) (Fig. 1A)
In FH-hTERT stably transfected with pbcatS33Y (FH-hTERT pbcatS33Y), increased b-catenin levels resulted in a robust upregulation in downstream pathway activity (Fig. 1C)
Summary
Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide with continuously rising incidence rates in Western countries [1, 2]. The principal clinical risk factors for hepatocellular carcinoma are well defined, molecular mechanisms contributing to tumor initiation and early progression are still not completely understood. In almost 90% of human malignancies, telomerase activation, characteristically mediated by reex-. Pression of the rate-limiting catalytic subunit human telomerase reverse transcriptase (hTERT), has been observed as early event [3]. Because of the end replication problem, telomeres progressively shorten with every cell division until a critical length is reached and the cells enter senescence, a postmitotic quiescent state [6]. On the basis of these findings, telomerase activation has been proposed as an early prerequisite in hepatocarcinogenesis [11, 12]. In addition to a limitless replicative potential (immortalization), further genetic alterations are required for self-sufficiency in growth signals, insensitivity to antigrowth signals, effective evasion of apoptosis, and altered differentiation [13]
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