Figure 6.

PHLDA1 Levels Regulate Sensitivity to Trastuzumab and Lapatinib Treatment

(A) Western blot analysis of PHLDA1 levels in MCF7/HER2-18 cells cultured with 1 μM trastuzumab or IgG control for 72 hr.

(B) MCF7/HER2-18 cell number following 3-day treatment with 1 μM trastuzumab preceded by 48-hr siRNA knockdown of PHLDA1 or scrambled control.

(C) In situ hybridization for PHLDA1 expression in MCF7/HER2-18 xenograft tumors. Four-week-old tumors from mice treated with an IgG control showed strong PHLDA1 mRNA expression (brown), whereas treatment with trastuzumab resulted in significantly weaker staining, as shown in graph on right. Sections were counterstained with hematoxylin, and dotted boxes represent zoomed-in areas. Data are presented as mean ± SEM from at least eight mice for each condition. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, compared with IgG controls.

(D) Western blot showing PHLDA1 levels in parental and lapatinib-resistant SKBR3 and HCC1954 cells treated with 2 μM lapatinib or DMSO control for 48 hr.

(E and G). Upper: H&E staining of SKBR3^LapR (E) and HCC1954^LapR cells (G) containing a doxycycline-inducible PHLDA1 expression construct. Cells were grown in mini-organotypic cultures for 7 days with or without 2 μM lapatinib and 1 μg/mL doxycycline. Lower: Ki67 staining with nuclei counterstained by DAPI. Right: quantitation of cell number and Ki67-positive nuclei. Data are presented as mean ± SEM. Images are representative of at least three independent experiments. H&E image scale bar, 100 μm; Ki67 image scale bar, 50 μm. ^∗∗∗p ≤ 0.001. H&E images are automatically spliced composites.

(F and H) Western blot showing PHLDA1 levels in parental and resistant SKBR3 cells (F) and HCC1954 cells (H) following treatment with doxycycline.