Suppression of autophagy by FIP200 deletion inhibits mammary tumorigenesis

Abstract

Autophagy is a conserved cellular process for bulk degradation of intracellular protein and organelles in lysosomes. In contrast to elegant studies of beclin1 using mouse models and cultured cells demonstrating a tumor suppression function for autophagy, knockout of other essential autophagy proteins such as ATG5, ATG7, or FIP200 (FAK family-interacting protein of 200 kDa) in various tissues did not lead to malignant tumor development in vivo. Here, we report that inhibition of autophagy by FIP200 ablation suppresses mammary tumor initiation and progression in a mouse model of breast cancer driven by the PyMT oncogene. Deletion of FIP200 resulted in multiple autophagy defects including accumulation of ubiquitinated protein aggregates and p62/SQSTM1, deficient LC3 conversion, and increased number of mitochondria with abnormal morphology in tumor cells. FIP200 deletion did not affect apoptosis of mammary tumor cells or Ras-transformed mouse embryonic fibroblasts (MEFs), but significantly reduced their proliferation in both systems. We also observed a reduced glycolysis and cyclin D1 expression in FIP200-null mammary tumor cells and transformed MEFs. In addition, gene profiling studies revealed significantly elevated expression of interferon (IFN)-responsive genes in the early tumors of FIP200 conditional knockout mice, which was accompanied by increased infiltration of effector T cells in the tumor microenvironment triggered by an increased production of chemokines including CXCL10 in FIP200-null tumor cells. Together, these data provide strong evidence for a protumorigenesis role of autophagy in oncogene-induced tumors in vivo and suggest FIP200 as a potential target for cancer therapy.

Figure 1.

Conditional deletion of FIP200 in MaEC suppresses breast cancer initiation, progression, and metastasis. (A) Kaplan-Meier analysis of mammary tumor development in the Ctrl-MT (n = 44), Cre-MT (n = 21), and CKO-MT (n = 35) mice. CKO-MT versus Ctrl-MT or Cre-MT: P < 0.01 by the log-rank test. (B) Scatter plots showing days of survival for indicated mouse strains before they succumb to their tumor burden. Horizontal line represents the mean. (C) Representative mammary gland whole mounts from 10-wk-old Ctrl-MT (left) and CKO-MT mice (right). The average mass of inguinal mammary glands from Ctrl-MT (left, n = 6) and CKO-MT (right, n = 7) mice are also shown. Bar, 5 mm. (**) P < 0.01. (D) Quantification of the area occupied by hyperplastic lesions expressed as a percentage of the total mammary gland surface. (E) Lysates were prepared from four tumors in four different Ctrl-MT (left four lanes) and CKO-MT (right four lanes) mice and analyzed by immunoblotting using antibodies against various proteins as indicated. (F,G) Lungs were harvested from Ctrl-MT (n = 17) and CKO-MT (n = 12) mice at 7 wk after initial detection of palpable primary tumors. (F) Representative images from these mice. (G) The mean ± SD of the number of metastatic nodules on the surfaces of lungs per mouse.

Figure 2.

Ablation of FIP200 leads to autophagic defects in mammary tumor cells. (A) Sections from mammary tumors of Ctrl-MT and CKO-MT mice were analyzed by immunohistochemistry using anti-ubiquitin (top panels) or anti-p62. Note the ubiquitin- and p62-positive aggregates in tumor cells from CKO-MT mice (red arrows, right panels), but not Ctrl-MT mice (left panels). Bars, 25 μm. (B) Mammary tumor sections of Ctrl-MT and CKO-MT mice were examined by transmission electron microscopy. Note the abnormal morphology of mitochondria in CKO-MT tumor cells (red arrows, right panel). Bar, 1 μm. (C,D) Mammary tumor cells from CKO-MT or Ctrl-MT mice were analyzed for total mitochondrial mass by MitoTracker Green staining (C) or for respiring mitochondria mass by MitoTracker Deep Red staining (D). (E) Mammary tumor cells from CKO-MT or Ctrl-MT mice were incubated in the complete or starvation medium (HBSS) for 1 h with or without 100 nM bafilomycin A1 (Baf), as indicated. The cell lysates were then prepared and analyzed by immunoblotting using anti-LC3 antibody. (F) Lysates were prepared from four tumors in four different Ctrl-MT (left four lanes) and CKO-MT (right four lanes) mice and analyzed by immunoblotting using antibodies against various proteins as indicated.

Figure 3.

Inactivation of FIP200 decreases proliferation of mammary tumor cells. (A,B) The apoptosis and proliferation of tumor cells in MIN lesions of Ctrl-MT and CKO-MT mice in vivo were measured by immunohistochemistry using anti-cleaved caspase 3 (A) and BrdU incorporation assays (B), respectively. Bars, 50 μm. Data on the right side graphs are mean ± SD (n = 4 for each sample) of relative numbers of cleaved caspase 3⁺ or BrdU⁺ cells in MIN lesions. (C–E) Mammary tumor cells isolated from Ctrl-MT and CKO-MT mice were measured for apoptosis using TUNEL assay (C), proliferation by BrdU incorporation assay (D), and anchorage-independent growth in soft agar (E). Data on the right side graphs are mean ± SD. Bars: C,D, 50 μm; E, 300 μm. (F,G) CKO-MT and Ctrl-MT tumor cells were analyzed for the level of glucose uptake (2-NBDG uptake) following 6 h of incubation (F) or for intracellular level of lactate in growth medium (G), as described in the Materials and Methods. (H) Lysates were prepared from mammary tumor cells isolated from Ctrl-MT and CKO-MT mice and analyzed by immunoblotting using antibodies against various proteins as indicated. (I) Tumor cells from FIP200^f/f;MMTV-PyMT mice were infected by recombinant adenoviruses encoding Cre (Ad-Cre) or a control insert (Ad-lacZ) to produce FIP200 KO and Ctrl tumor cells, respectively. These tumor cells were then infected with recombinant retroviruses encoding HA-tagged cyclin D1 (cyclin D1) or control viruses (Mock) to prepare stable pools of the infected cells. After 6 d of incubation in growth medium, the cells were counted, and the mean ± SD of relative growth is shown. The inset shows expression of HA-tagged ectopic cyclin D1 in KO cells (right lane) but not vector-infected Ctrl cells (left lane) or KO cells (middle line).

Figure 4.

Analysis of FIP200 deletion in Ras-transformed primary MEFs. (A) Primary MEFs from FIP200 floxed mice were infected with retroviral vectors encoding RFP-E1A and GFP-Ha-RasV12. After staining with DAPI, the cells were viewed under a fluorescent microscope for the expression of E1A and RasV12 (left) and nuclei (right). Bar, 50 μm. (B–F) RFP-E1A/GFP-RasV12-transformed primary MEFs from FIP200 floxed mice were infected with recombinant adenoviruses encoding Cre (Ad-Cre) or a control insert (Ad-lacZ) to produce FIP200 KO (FIP200^−/−) and control (FIP200^f/f) cells, respectively. (B) Lysates from the cells were analyzed by immunoblotting using antibodies against FIP200 or actin as indicated. The cells were measured for multiplication (C), apoptosis using TUNEL assay (D), proliferation by BrdU incorporation assay (E), and anchorage-independent growth in soft agar (F). Data in C–F are mean ± SD. (G) FIP200 KO (FIP200^−/−) and control (FIP200^f/f) cells were grown in medium containing the indicated concentration of serum for 24 h. Cell lysates were then prepared and analyzed by immunoblotting using antibodies against various proteins as indicated. (H) FIP200 KO (FIP200^−/−) and control (FIP200^f/f) cells were incubated in growth medium in the presence or absence (Mock) of rapamycin (100 nM), as indicated. After 24 h, the cells were counted, and the mean ± SD of relative growth is shown. (I) FIP200 KO cells were infected by recombinant retroviruses encoding HA-tagged cyclin D1 (cyclin D1) or control viruses (Mock) to prepare stable pools of the infected cells. After 3 d of incubation in growth medium, the cells were counted, and the mean ± SD of relative growth is shown. (Inset) Expression of ectopic cyclin D1 in cyclin D1 cells (right lane) but not Mock cells (left lane).

Figure 5.

Global changes in gene expression profile of mammary tumors from CKO-MT mice. (A,B) Heat map of genes showing differential up-regulation (A) or down-regulation (B) of at least twofold in tumors from CKO-MT mice compared with those from Crtl-MT mice. Representative up-regulated and down-regulated genes are marked on the right. (C,D) A select group of up-regulated genes were validated using qRT–PCR. The mean ± SD (n = 3) of relative levels (normalized to Ctrl-MT tumors) from three independent experiments is shown.

Figure 6.

Analysis of immune responsive genes and infiltration of effector T cells in mammary tumors of CKO-MT mice. (A) Mammary tumor cells were isolated from Ctrl-MT and CKO-MT mice. They were cultured in vitro and then treated with 10 ng/mL IFN-α or IFN-γ, or media mock as a control, as indicated. The expression levels of several IFN-responsible genes were analyzed by qRT–PCR. The mean ± SD of relative levels (normalized to mock Ctrl-MT tumor cells) is shown. (B) Cryosections of mammary tumors from Ctrl-MT (left) and CKO-MT (right) mice were immunostained with antibody against CD45 (top) or CD8 (bottom) to detect infiltrated leukocytes (shown by green FITC fluorescence). Nuclei were counterstained with DAPI (blue). Bar, 100 μm. (C) Freshly isolated immune cells from mammary tumors in Ctrl-MT and CKO-MT mice were analyzed by flow cytometry for CD45⁺ leukocytes. (D,E) Freshly isolated immune cells from mammary tumors in Ctrl-MT and CKO-MT mice were analyzed for subpopulations of T cells by flow cytometry. CD3 gated T cells were measured for the fraction of CD8⁺IFN-γ⁺ and CD4⁺IFN-γ⁺ cells (D) or CD4⁺Foxp3⁺ regulatory T cells (E). (F) Kaplan-Meier analysis of mammary tumor initiation in CD8⁺ cells depleted of CKO-MT (n = 7) and IgG control-treated CKO-MT (n = 7) mice. P < 0.01 by the log-rank test. (G,H) Mammary tumor cells from Ctrl-MT or CKO-MT mice were incubated in growth medium in the absence (Mock) or presence of IFN-γ (1 ng/mL or 20 ng/mL), as indicated. (G) Representative image of CKO-MT tumor cells incubated in Mock or 20 ng/mL IFN-γ after 3 d. Bar, 25 μm. (H) The cells were counted after 6 d of incubation, and the mean ± SD of the number of cells (5 × 10⁴ at day 0) is shown. (**) P < 0.01.

Figure 7.

Increased chemokine production in FIP200-null tumor cells and a working model. (A) Mammary tumor cells were isolated from Ctrl-MT or CKO-MT mice. RNA was isolated from the purified cells and subjected to analysis by qRT–PCR to detect the expression of various chemokines as indicated. The mean ± SD of relative levels (normalized to Ctrl-MT tumors) from three independent experiments is shown. (*) P < 0.05; (**) P < 0.01. (B,C) Mammary tumor cells from Ctrl-MT or CKO-MT mice (B) and FIP200 KO (FIP200^−/−) or control (FIP200^f/f) cells (C) were transfected with 10 μg/mL poly(I:C) or mock using Lipofectamine. RNA was isolated at 8 h after stimulation and subjected to qRT–PCR analysis for the relative level of IFN-β (left) and CXCL10 (right). (**) P < 0.01. (D) Mammary tumor cells from Ctrl-MT or CKO-MT mice were stimulated by poly(I:C) for 8 h as described in B. The amount of CXCL10 in the medium was then measured by ELISA using a kit (R & D Systems). (**) P < 0.01. (E) A working model summarizing the potential mechanisms of suppression of mammary tumorigenesis and progression in CKO-MT mice. Inactivation of FIP200 results in defective autophagy in tumor cells that may trigger multiple events leading to the inhibition by mammary tumorigenesis through both reduced cell proliferation and increased immune surveillance in the host.