Part:BBa_K4469003

a2bM-alpha fetoprotein (AFP) promoter

a2bM-AFP is composed of 2 enhancers A (EA), enhancer B (EB­), and hepatocellular carcinoma (HCC) specific alpha-fetoprotein (AFP) promoter.

AFP is a glycoprotein produced by the visceral endoderm of the yolk sac during the embryonic period, and under normal physiology, it should remain at low levels throughout the lifespan. However, the re-expression of AFP in patients with HCC has been observed in 50%-70% of HCC patients. Currently, AFP 400 ng/mL is used as a diagnostic threshold for liver cancer (Bialecki & di Bisceglie, 2005). Among patients of TNM III/IV, over half have an AFP level of 21-400 ng/mL or more (Galle et al., 2019). At the early stage of hepatocarcinogenesis, the AFP gene is reactivated in liver cells. Cytoplasmic AFP promotes malignant liver cells proliferation via induction of Src and c-myc expression. Later, metastasis of liver cancer is further promoted by stimulating the expression of metastasis-related genes (e.g., K19 and EpCAM) (Lu et al., 2016). AFP can also act as a pro-proliferative protein. By activating the cAMP-protein kinase A pathway, Ca2+ channels open and increase calcium influx, thus DNA synthesis and tumor proliferation (Hu et al., 2022).

The specificity of this AFP promoter variant is assessed by the AFP expression level in various cell types: AFP-positive HCC (Huh7, HepG2, and Hep3B), AFP-negative HCC (HepI), non-HCC cancer cells (U343 and A549), and normal fibroblasts (BJ, W138, and IMR90). a2bM selectively induced expression of luciferase in all AFP-positive HCC cells, while minimal to no luciferase activity is observed in AFP-negative cells and normal cells. This shows that the promoter variant can express genes specifically in AFP-positive cancer cells, minimizing the off-target effects on functional hepatocytes. The addition of EA and EB helps to further augment AFP activity by increasing the number of binding sites for liver-enriched transcription factors, such as miRNA-122, that are also overexpressed in HCC. Moreover, this promoter demonstrates a more robust induction of transgene expression than entire-length human AFP. This could be due to the removal of the silencer in the a2bM variant, which helped enhance the transcriptional activity of AFP promoter without compromising its specificity. In AFP-positive HCCs, transcription would be activated, and transgenes are shown to be expressed 43- to 456-fold higher than in cells with AFP promoter lacking the enhancers (Yoon et al., 2018). Therefore, a2bM-AFP is inserted into the vector to allow HCC-specific transcription and enhance transgene expression in AFP-positive HCC.

Reference

Bialecki, E. S., & di Bisceglie, A. M. (2005). Diagnosis of hepatocellular carcinoma. HPB, 7(1), 26–34. https://doi.org/10.1080/13651820410024049

Galle, P. R., Foerster, F., Kudo, M., Chan, S. L., Llovet, J. M., Qin, S., Schelman, W. R., Chintharlapalli, S., Abada, P. B., Sherman, M., & Zhu, A. X. (2019). Biology and significance of alpha-fetoprotein in hepatocellular carcinoma. Liver international : official journal of the International Association for the Study of the Liver, 39(12), 2214–2229. https://doi.org/10.1111/liv.14223

Hu, X., Chen, R., Wei, Q., & Xu, X. (2022). The Landscape Of Alpha Fetoprotein In Hepatocellular Carcinoma: Where Are We? International Journal of Biological Sciences, 18(2), 536–551. https://doi.org/10.7150/ijbs.64537

Lu, Y., Zhu, M., Li, W., Lin, B., Dong, X., Chen, Y., Xie, X., Guo, J., & Li, M. (2016). Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells. Journal of Cellular and Molecular Medicine, 20(3), 549–558. https://doi.org/10.1111/jcmm.12745

Yoon, A. R., Hong, J., Kim, M., & Yun, C. O. (2018). Hepatocellular carcinoma-targeting oncolytic adenovirus overcomes hypoxic tumor microenvironment and effectively disperses through both central and peripheral tumor regions. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-20268-6