(A) Significant downregulation in AHRR mRNA (between 30% and 90%) was observed in tumors from several origins. Data are presented as a ratio of mRNA levels in tumor versus normal control. (B) Primary colon tumors showed a very strong downregulation of AHRR mRNA when compared with normal controls. Interestingly, nonmalignant colon polyps exhibited a more moderate downregulation of AHRR mRNA when compared with colon tumors. Statistically significant differences were achieved when comparing levels of AHRR in normal tissue with levels in polyps. (C) AHRR expression levels in normal cervical tissue, cervical tumor cell lines, and primary cervical tumors. Strong downregulation or complete ablation of AHRR mRNA was observed in 100% of cell lines and 80% of the primary tumors when compared with normal controls. Little variation was observed in the levels of AHRR mRNA among the normal controls. (D) All ovarian tumor cell lines included in this study showed downregulation of AHRR mRNA levels when compared with normal ovarian cell lines. (E) Box-and-whisker plot showing significant downregulation AHRR mRNA levels observed in lung tumors and normal tissues from the same patients. ***P < 0.001.

(A) A549E (black bars), A549F (white bars), and A549G (gray bars) were incubated in serum-free media (R0) or the appropriate media containing α-Fas or MK886. In all experiments (n = 3), A549F/G showed enhanced resistant to proapoptotic signals when compared with A549E. (B) DIVAA analysis showed that A549F and A549G have enhanced angiogenic potential when compared with A549E, and this effect was more prominent for A549G than for A549F (n = 8). (C) Silencing of AHRR (A549F/G) enhances the migratory (black bars) and invasive (white bars) potential of the A549 tumor cell line (n = 6). Detail photos of the porous membranes used to determine migratory potential of A549E and A549G are also shown. Original magnification, ×10. *P < 0.05; **P < 0.01; ***P < 0.001.

(A) In normal cells, activation of AHR causes induction of AHRR through XREs present in its promoter. AHRR acts as a negative regulator of AHR by competing for binding to ARNT and XREs present in the promoter regulatory region of variety of genes. The balanced relationship between the positive and negative transactivation signals of AHR and AHRR results in cellular homeostasis. (B) In cancer cells, methylation of the AHRR promoter and LOH cause blockade of its expression despite the higher levels of AHR. Thus absence of AHRR eliminates competition for binding to ARNT and XREs, which results in an imbalance between positive and negative transactivation signals, thereby causing the induction of a battery of genes related to tumorigenesis and cancer progression.

(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.

(A) Overexpression of AHRR partially blocked expression of AHR-induced genes such as CYP1A1 (plasmid amounts expressed in μg). (B) Increased levels of AHRR in A549 resulted in reduced anchorage-dependent growth. The blue line represents A549-AHRR, and the red line represents A549-empty. Dotted lines denote standard deviation (n = 4). The maximum difference was reached 56 hours after inoculation of cells in the wells. (C) Ectopic expression of AHRR resulted in reduced anchorage-independent growth (adjacent images). Original magnification, ×10 (C); ×2 (D). (D) Representative images of tube formation assays using cocultures of A549-empty or A549-AHRR and PAE-GFP cells. A549-empty induced tube formation of PAE-GFP cells comparable to that of the internal positive control (10% FBS). Reduced tube formation was observed when PAE-GFP cells were cocultured with A549-AHRR. **P < 0.01; ***P < 0.001.