Figure 5.

Malignant growth of MDA‐MB‐231 cells is mediated by PKN3. (A) PKN3 and RhoC expression levels in breast cancer cell lines. Lysates from the indicated breast cancer lines grown in the presence or absence of serum were analyzed for expression and activation of PKN3 as well as for RhoC levels by immunoblotting; p110α served as a loading control. Subsequent experiments were carried out in MDA‐MB‐231 cells (underlined). (B) Stably transduced MDA‐MB‐231 cells with Dox‐dependent expression of either PKN3 or p110β shRNAs were analyzed in parallel with samples expressing an unrelated control shRNA. Cells were grown with or without 250ng/ml Dox for 72h and extracts were immunoblotted with antibodies against PKN3, p110β and phospho‐Akt (T308). Akt served as a loading control. (C) The engineered cells were pre‐treated with or without Dox for 72h, embedded in 80% Matrigel (11–12mg/ml) and photographed after 7 days at 10× magnification. Medium was replaced every 2–3 days ±Dox. (D) In parallel, cells were cultured as mammospheres in low binding plates with or without Dox. Representative spheroids from mock or Dox‐treated plates were photographed after 4 days at 5× magnification (left). Comparable size spheroids were selected and cultured individually in 24‐wells and photographed on days 9 and 13 (right). (E) Stable PKN3 (orange bars), p110β (blue bars) or control (white bars) shRNA expressing cells were transplanted into the mammary fat pad of female mice. The animals were split into two groups, one group was treated with Dox to induce shRNA expression and the second group was mock treated; with 15 animals per group (n). Primary mammary tumors were measured weekly for 6 weeks, and on day 42 the animals were killed and evaluated. Each bar represents the mean tumor volume±S.E.