Extended Data Fig. 8: RAP2 deletion contributes to aberrant acinus growth and tumorigenesis of MCF10A cells in a YAP- and TAZ-dependent manner.
a, Representative images showing soft-agar assays of MCF10A cells. Overexpression of the constitutively active YAP(5SA) (mutation of all five LATS1- and LATS2-phosphorylation serines to alanines in YAP) strongly promotes anchorage-independent growth. Combined deletion of RAP2, MST1 and MST2, but not either group alone, also causes anchorage-independent growth of MCF10A cells. Images are representative of three biologically independent experiments with similar results. Scale bar, 35 mm. b, RAP2 inhibits tumorigenicity of MST1/2-dKO MCF10A cells. MST1/2-dKO and RAP2-MST1/2-KO MCF10A cells were injected into NOD/SCID mice. Tumour weight on day 32 after injection is presented as mean ± s.e.m. n = 6 biologically independent xenografts, ****P = 0.000025, two-tailed t-test. c, Representative tumour sizes. Only three very small xenografts were recovered from the initial 6 subcutaneous injections for MST1/2-dKO cells. Images representative of six biologically independent xenografts for each group that were initially made in the NOD/SCID mice. d, Immunohistochemistry staining with an antibody recognizing human HLA Class I. Only the acinus structures in the xenografts were formed by MCF10A cells. Stroma cells negative for HLA class I were derived from the host mice. Images are representative of two biologically independent experiments with similar results. Scale bars, 50 µm. e, Haematoxylin and eosin staining of xenografts from MST1/2-dKO MCF10A cells shows that largely hypocellular connective tissue is observed as stroma from the host animals. By contrast, RAP2-MST1/2-KO xenografts showed MCF10A cell-derived acinar and duct structures exhibiting nuclear polymorphisms, irregular nuclear contour, hyperchromasia, prominent nucleoli (star), and pathological mitosis (arrows). Images representative of two biologically independent experiments with similar results. Scale bars, 50 µm. f, Western blot showing knockdown of YAP or TAZ by lentiviral shRNAs in RAP2-MST1/2-KO MCF10A cells. shYAP#2 and shTAZ#1 were used for the xenograft studies. The image is representative of two independent experiments with similar results. g, Knockdown of YAP and TAZ inhibits tumour growth of RAP2-MST1/2-KO MCF10A cells. Tumour weight on day 32 is presented as mean ± s.e.m. Two-tailed t-test, n = 6, ****P = 0.000010. h, Xenografts from NOD/SCID mice, in which six biologically independent xenografts were generated for each group. i, Western blot showing that MCF10A-T cells are generated by expression of the oncogenic mutant H-RAS-G12V. H-RAS-G12V expression activates ERK whereas RAP2 deletion has no effect on ERK. The comparable phosphorylation levels of ERK1 and ERK2 in wild-type and RAP2-KO MCF10A-T cells indicate that the difference in xenograft growth was not due to difference in ERK1 and ERK2 activity. Image representative of two independent experiments with similar results. j, MCF10A and MCF10A-T xenografts from nude mice, in which six biologically independent xenografts were generated for each group and yielded similar results.
