Figure 2. RASAL2 functions as a tumor suppressor in breast cancer.

(A) Growth curves of MDA-MB-361 and MCF7 cells expressing RASAL2 or LacZ. Data points show triplicate averages ± SD. There were no statistically significant differences in proliferation. Western blot on right confirms ectopic RASAL2 expression.

(B) Soft agar colony formation of MCF7, BT474, MDA-MB-361, and SUM159PT cells expressing RASAL2 or LacZ. Data show relative number of colonies ± SD. There was a statistically significant decrease in anchorage-independent growth upon ectopic RASAL2 expression in RAS wild type cell lines (MCF7 and BT474 p<0.0001; MDA-MB-361 p=0.002) but not in the HRAS mutant cell line SUM159PT. Western blots confirm ectopic RASAL2 expression.

(C) Xenograft tumor formation of MDA-MB-361 and MDA-MB-231 cells expressing RASAL2 or LacZ. MDA-MB-361 cells were injected orthotopically into female NOD/SCID mice; MDA-MB-231 cells were injected subcutaneously into female nude mice. Horizontal bars indicate mean tumor volume. There was a statistically significant decrease in tumor growth upon ectopic RASAL2 expression (p<0.0001) in the RAS wild type cell line MDA-MB-361 but not in the KRAS mutant cell line MDA-MB-231. Western blots below confirm ectopic RASAL2 expression.

(D) Xenograft tumor formation of CAMA1 cells infected with shRNAs targeting RASAL2 or non-targeting control shRNA and injected subcutaneously into female NOD/SCID mice. Horizontal bars indicate mean tumor volume. There was a statistically significant increase in tumor growth upon RASAL2 inactivation (p=0.0007). Western blot confirms RASAL2 knockdown.