Ras- and PI3K-dependent breast tumorigenesis in mice and humans requires focal adhesion kinase signaling

Abstract

Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland-specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin beta4-dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130Cas. These results suggest that FAK supports Ras- and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.

Figure 1. Amplification of FAK in human breast cancer.

(A–D) Primary tumors (n = 79) from the MSKCC breast tumor collection were subjected to FISH and DNA microarray analysis. (A) Average number of copies of FAK and CEP8. (B) FAK/CEP8 ratio in the same tumors. Tumors are ordered as in A. (C) Images from representative cases: not amplified (left), polysomy and amplification (middle), and amplification (right). Original magnification, ×630; ×945 (insets). (D) Correlation between FAK copy number and mRNA expression. r² = 0.16; P = 0.0002. (E) Kaplan-Meier analysis of metastasis-free survival in the NKI cohort. n = 295; P = 0.0062.

Figure 2. Conditional deletion of FAK suppresses mammary tumorigenesis in MMTV-PyMT mice.

(A) Kaplan-Meier analysis of tumor onset in mice of the indicated genotypes. P < 0.0001, FAK^+/+ or FAK^+/fl versus FAK^fl/fl. (B) Mean number of tumors per mouse (±SEM) in MMTV-PyMT mice of the indicated genotype at the time of sacrifice. P < 0.0001, FAK^+/+ or FAK^+/fl versus FAK^fl/fl. (C) Whole mounts of mammary glands from 12-week-old MMTV-PyMT mice of the indicated genotype were stained with hematoxylin. Percentages indicate the frequency of observed phenotypes (n = 8 per genotype). Original magnification, ×20. (D) Mammary glands from 6-month-old MMTV-PyMT/Cre;FAK^fl/fl;R26R mice were subjected to β-galactosidase staining and counterstaining with eosin. Insert in top left panel shows early MIN lesions budding out of normal ducts (black arrows) and an unaffected duct (green arrow). Graph on the bottom left indicates the percentage of X-gal–positive and –negative MIN lesions (error bars denote SEM); P = 2.8 × 10^–15. Graph on the bottom right indicates the percentage of FAK-null and FAK-expressing adenocarcinomas. Original magnification, ×200 (left); ×400 (inset); ×100 (right). (E) FAK and Cre staining on early MIN lesions from MMTV-PyMT;MMTV-Cre;FAK^fl/fl mice. The dotted outline indicates a FAK-positive early intraductal lesion. (F) Percentage of Ki67-positive tumor cells (error bars denote SEM) in FAK-expressing and FAK-null MIN lesions; P = 3.4 × 10^–11.

Figure 3. Deletion of FAK inhibits PyMT-transformed mammary tumor cells.

(A–F) Primary tumor cells from MMTV-PyMT;FAK^fl/fl mice were transduced with adeno-GFP or adeno-Cre, cultured in SFM or complete medium (FCS), and subjected to immunoblotting (A); cultured on collagen I, starved for 24 hours, and incubated in the presence of BrdU in complete medium for 24 hours (B); plated on collagen I and subjected to TUNEL assay (C); suspended in complete medium containing 0.5% methylcellulose and 1% BSA for 8 hours at 37°C and subjected to TUNEL assay (D); subjected to Matrigel invasion assay for 8 hours (E); or injected at 2 × 10⁵ in the mammary fat pads of NOD/SCID mice to evaluate tumorigenicity (F). The graphs indicate mean values ± SEM. (G–J). Primary tumor cells from MMTV-PyMT;FAK^fl/fl mice were transduced with empty retroviral vector or the indicated constructs, infected with adeno-GFP or adeno-Cre, subjected to immunoblotting (G), and injected orthotopically into NOD/SCID mice (H–J). (H) Mean tumor volumes (±SEM) over time (n = 4 per group). (I) Size of individual tumors and mean size in each class at day 39. (J) Tumor lysates were subjected to immunoblotting. FAK^–/– mammary epithelial cells were used as control. Ad tumor, adeno-GFP tumor. Error bars denote SEM.

Figure 4. Deletion of FAK suppresses metastasis to the lung.

(A) Tumor cells from MMTV-PyMT;FAK^fl/fl mice were transduced with adeno-GFP (Ad) or adeno-Cre (Ad-Cre) and TGL, injected in the tail vein of NOD/SCID mice, and subjected to bioluminescence imaging. The graph shows the normalized photon flux in arbitrary units (±SEM); P = 0.039. (B) Representative images of mice injected with tumor cells transduced with the indicated viruses. Scale indicates the dynamic range of luminescent signal. (C) Quantification of GFP⁺ tumor cells present on lung sections at days 1 and 2 after tail vein injection (top). The percentage of GFP⁺ tumor cells undergoing apoptosis was estimated by staining with antibodies against cleaved caspase-3 (bottom). ***P < 0.001; n = 4 per group. Error bars denote SEM. (D) Lung sections from mice injected with tumor cells transduced with the indicated viruses were stained with anti–PECAM-1 (red) and subjected to confocal analysis. Panels show representative images at days 1 and 2. Original magnification, ×630.

Figure 5. FAK kinase activity and Cas-binding motif of FAK promote mammary tumorigenesis.

(A–D) Primary tumor cells from MMTV-PyMT;FAK^fl/fl mice were transduced with empty retrovirus or the indicated FAK constructs, infected with adeno-GFP or adeno-Cre, and subjected to immunoblotting (A); synchronized in G₀ and subjected to BrdU incorporation assay (B); cultured in suspension for 8 hours and subjected to TUNEL assay (C); or subjected to Matrigel invasion assay for 8 hours (D). The graph in D indicates the mean number of invasive cells (±SEM) per microscopic field. *P < 0.05, **P < 0.01, ***P < 0.001 compared with adeno-GFP; n = 3. PyMT FAK-proficient cells were transfected with either GFP or 1 or 2 μg of CasΔSD constructs, synchronized in G₀, and subjected to BrdU incorporation assay (E). The graph depicts percentages of GFP⁺BrdU⁺ cells (±SEM).

Figure 6. FAK promotes Ras-mediated mammary tumorigenesis and cooperates with integrin β4 to sustain ErbB2-mediated tumorigenesis.

(A–C) Normal murine mammary gland (NMuMG) cells were transduced with empty vector (c) or vectors encoding shRNAs targeting murine FAK (1, 2) and subjected to immunoblotting (A); BrdU incorporation assay (B); and cultured under standard conditions (left) or resuspended for 8 hours (right) and subjected to TUNEL assay (C). (D–H) PyMT-, Ras-, and Neu-transformed mammary tumor cells expressing wild-type β4 (Neu) or signaling-defective β4 (Neu-β4-1355T) were transduced with empty vector or vectors encoding shRNAs targeting murine FAK (1, 2) and subjected to BrdU incorporation assay (D); cultured for 6 hours and subjected to TUNEL assay (E); or subjected to Matrigel invasion assay for 8 hours (F). Ras-transformed cells transduced with the indicated viruses were stained with X-gal (blue) (G). Original magnification, ×100. (H) Control and FAK-silenced tumor cells were subjected to immunoblotting; the asterisk indicates JunD phosphorylated at Ser100. Graphs indicate mean values (±SEM). *P < 0.05, **P < 0.01, ***P < 0.001; n = 3.

Figure 7. FAK signaling through p130^Cas sustains human breast cancer cells carrying clinically prevalent oncogenic mutations.

(A) The indicated human breast cancer cells were infected with control virus or shRNAs 1 and 6 targeting FAK, synchronized in G₀, and incubated in the presence of BrdU in complete medium for 24 hours. The graph indicates the percentage of BrdU⁺ cells (±SEM). n = 3. Oncogenic mutations and mean percentage of inhibition after FAK silencing are indicated below. (B) Cancer cells were transfected with control or siRNA oligonucleotides targeting human p130^Cas (s1, s2) and subjected to immunoblotting or seeded in microtiter wells, cultured for 4 days in complete medium, fixed, and stained with crystal violet. (C) Cancer cells transfected with the indicated siRNAs were counted 4 days after seeding. Control values were normalized to 100%. ***P < 0.001. Error bars denote SEM.

Figure 8. Loss of FAK induces senescence in tumor cells transformed by Ras.

(A–D) MDA-MB231 cells were transduced with control virus or vectors encoding shRNAs targeting FAK, cultured over a period of 4 days, and photographed at the indicated times (A; original magnification, ×100); stained with X-gal on day 4 (B; original magnification, ×100); subjected to immunoblotting with the indicated antibodies (C); or stained with DAPI alone (top) or antibodies against trimethylated H3 K9 (H3 K9triM), followed by counterstaining with DAPI (bottom) to visualize SAHF (D; original magnification, ×630; ×1,260 (insets).