Figure 6. Gli2 is required to increase PTHrP expression, bony invasion and bone destruction.

A. Loss of Gli2 using shRNA decreases bone destruction in vivo. CAL27 cells stably expressing shRNA against a non-silencing control sequence or Gli2 were injected into the masseter muscle of male athymic mice and allowed to progress for eight weeks. Mice were then sacrificed and mandibles dissected for high resolution x-rays. Significantly less bone destruction, as measured by lesion area, was observed in the shGli2 group. B. Loss of Gli2 decreases PTHrP levels in vivo. CAL27 tumors expressing shRNA against Gli2 show significantly lower PTHrP protein levels by IHC. (Inset is 40X with 200μm scale bars. Arrows denote positive staining) Loss of Gli2 expression is verified using IHC. C. Loss of Gli2 decreases osteoclasts numbers in vivo. Tartrate Resistant Acid Phosphate (TRAP) staining was used to identify osteoclasts at the tumor bone interface of mandible sections. We found a significantly larger number of TRAP-positive multinucleated cells in the control group as compared to the shGli2 group. D. Gli2 mRNA is associated with bony invasion in clinical samples. qRT-PCR was done on human OSCC clinical samples, where patients that underwent a mandiblectomy were classified as bony invasive (BI), while samples from patients who did not undergo amandiblectomy were classified as non-bony invasive (NBI). Samples from the BI group had significantly higher expression of Gli2. E. Gli2 protein correlates with bony invasion in human clinical samples. A larger cohort of clinical samples based on the selection criteria described above was used for IHC against Gli2. We show that Gli2 protein also significantly correlated with bony invasion.