Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion in vitro To investigate the contribution Nutlin-3a buy of p21 to tumor formation and progression in breast cancer, we used a bone meta static cell line SCP2, a sub progeny of the human triple negative breast cancer MDA MB231 cells. We first assessed the effect of suppres sing p21 on tumor growth using a mammary fat pad xeno graft mouse model. A specific p21 shRNA was stably transfected to generate a pool of p21 deficient SCP2 cells. Knockdown of p21 using shRNA efficiently reduced p21 protein expression, as compared to parental SCP2 cells. Parental and shRNA p21 SCP2 cells were orthotopically injected into the mammary fat pad of female Balb/c nude mice. Tumor growth was monitored weekly.

There was no difference in the rate of primary tumor formation or tumor size between animals injected with parental or p21 deficient cells, suggesting p21 is not likely involved in tumor formation. Next, we evaluated the effect of p21 depletion on tumor invasiveness, a critical step for early tumor progression. Intact tumors were taken with the overlaying skin and surrounding deep tissues and analyzed by a pathologist. Tumor invasiveness was assessed by determining the extent of infiltration of cancer cells to the surrounding tissue, as previously described. As shown in Figure 2C, tumors from the parental SCP2 group dis played no clear margin with the surrounding tissues and were deeply invading into nearby structures.

In contrast, tumors derived from animals transplanted with p21 depleted SCP2 cells formed a well encapsulated tumor mass that did not invade the surrounding tissues, strongly suggesting that p21 plays an important role in tumor invasion. This was confirmed in vitro, as p21 gene silencing in SCP2 cells inhibited both cell migration and invasion. As shown in Figure S2A, none of the animals in which parental or p21 depleted SCP2 cells were injected into the mammary fat pad developed any bone lesions after two months, the date at which mice had to be sacrificed due to the tumor size. This timing may have been insufficient for tumor cells to grow into visible distant lesions in the mouse. Thus, to investigate whether p21 is involved in the later stage of breast cancer progression, we examined its involvement in the development of bone osteolytic lesions using an intratibia injection model of parental and p21 deficient SCP2 cells in female Balb/c nude mice. By by passing the early steps of metastasis, this experi mental model allows for the assessment of tumor cell metastasis and survival in the bone Anacetrapib marrow.