The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers
Enrique Zudaire, … , Michael Birrer, Frank Cuttitta
Enrique Zudaire, … , Michael Birrer, Frank Cuttitta
Published January 2, 2008
Citation Information: J Clin Invest. 2008;118(2):640-650. https://doi.org/10.1172/JCI30024.
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Research Article Oncology

The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers

Abstract

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung cancer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers.

Authors

Enrique Zudaire, Natalia Cuesta, Vundavalli Murty, Karen Woodson, Lisa Adams, Nieves Gonzalez, Alfredo Martínez, Gopeshwar Narayan, Ilan Kirsch, Wilbur Franklin, Fred Hirsch, Michael Birrer, Frank Cuttitta

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Figure 2

Effects of siRNA-induced silencing of AHRR on growth.

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Effects of siRNA-induced silencing of AHRR on growth. (A...
(A) On a time-course MTT assay, A549G (squares) grew 4.3-fold and A549F (triangles) grew 2.6-fold faster than the empty nonsilenced A549 cells (circles, A549E; diamonds, A549SR). No differences were observed between A549E and A549SR. (B–D) Cells deficient in AHRR (A549F [C] and A549G [D]) showed a significant shift in the number of cells towards S and G2/M phases as compared with the control A549E (B). Consistently, a reduction in the number of cells in G0/G1 phase was observed in A549F/G. (E and F) Artificial downregulation of AHRR enhanced A549 (E) and MCF10A (F) colony formation (60% and 300% increase in average colonies for A549F/G and A549E, respectively). MCF10A-F and MCF10A-G were able to clone in soft agar, while, as expected, MCF10A-E did not form colonies. All clonogenic assays were run in triplicate. (G and H) Comparison of the morphology of the colonies formed by A549E (G) and A549G (H). A549E grew in compact spheroids with defined contours. A549G colonies showed irregular morphology and cells detached and partially separated from the core of the colony. Original magnification, ×20. (I) In an in vivo experiment, A549 cells transfected with a siRNA for AHRR showed a significant increase in xenograft tumor volume 8 weeks after injection when compared with empty plasmid–transfected A549 cells (3-fold increase for A549F [squares] compared with A549E [triangles] and 7-fold increase for A549G [diamonds] compared with A549E). n = 10 animals/group. *P < 0.05; **P < 0.01; ***P < 0.001.