Abstract #3778: The analysis of activin receptor-like kinase 1 (ALK1) regulatory fragment which regulates tumor feeding artery-specific expression.
Authors: Yoshikuni Yonenaga, Rumi Suzuki, Fei Lan, Tsugio Seki
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Purpose: In order to utilize tumor feeding artery-specific induction of activin receptor-like kinase 1 (ALK1) in gene therapy, we characterized its regulatory fragment in this study. We previously showed with Alk1 (+/lacZ) reporter knock-in mice that Alk1 is not expressed in most tissues in adults and that it is specifically induced in arteries feeding angiogenesis-dependant lesions such as wound healing. It occurred to us that this gene might be exploited to aid in feeding artery-specific gene induction and delivery of a therapeutic peptide to another angiogenesis-dependent lesion, malignant tumors. We previously isolated a 9.2kb mouse Alk1 regulatory fragment. This fragment is, however, too large to be utilized in many of the viral vectors. In this study, we first examined Alk1 expression pattern in tumor angiogenesis, and next shortened and characterized its regulatory elements in transgenic (Tg) mice and adenoviruses. Methods and Results: MC38 cells were injected subcutaneously into Alk1 (+/lacZ) mice. Some feeding arteries started to be lacZ positive at the stage of palpable tumors. As tumor grew, lacZ-positive feeding arteries were increasing in number and caliber. Next, we generated a shortened 4.8kb Alk1 regulatory fragment, Alk1PIB, based on evolutionarily conserved region (ECR) analysis and created Tg mouse lines carrying the lacZ gene under this fragment. Similarly to MC38 tumor in Alk1 (+/lacZ) mice , B16 subcutaneous tumor in these Tg mice showed lacZ activity in feeding arteries, and the number of the positive arteries increased as the size of the tumor became bigger. We then examined the Alk1PIB regulatory activity in a recombinant adenovirus carrying the ZsGreen1-DR (ZsG) reporter gene. The virus was infected to human iliac arterial endothelial cells (HIAECs) and immortalized fibroblasts. No detectable ZsG expression was observed in fibroblasts. The Alk1PIB induced ZsG expression in 17.0% of HIAECs, which was comparable level to the hFLT1 (2.3%) and the hICAM2 (28.2%). To identify essential regulatory element in Alk1PIB fragment, we further utilized ECR analysis and found that a 152bp sequence (coreECR) in an intron was highly conserved in 11 mammals. The coreECR-deleted Alk1PIB(d1)-ZsG adenovirus showed significantly reduced reporter gene expression in HIAECs, indicating an enhancer function of this sequence. Conclusions: This fragment will be an invaluable tool in gene therapy to target arteries feeding tumors. For example, this activity may be utilized to express and secrete cytotoxic peptides in feeding arteries, which is then directly distributed to the downstream tumor tissues. It also may be utilized to express apoptosis-inducing gene to destroy the arteries and terminate blood supply to the tumor. Further analysis may enable us to enhance its induction capacity and specificity.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3778.