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. 2009 Jan 15;69(2):466-74.
doi: 10.1158/0008-5472.CAN-08-3078.

Mammary epithelial-specific ablation of the focal adhesion kinase suppresses mammary tumorigenesis by affecting mammary cancer stem/progenitor cells

Ming Luo  1 , Huaping Fan, Tamas Nagy, Huijun Wei, Chenran Wang, Suling Liu, Max S Wicha, Jun-Lin Guan
Affiliations

Mammary epithelial-specific ablation of the focal adhesion kinase suppresses mammary tumorigenesis by affecting mammary cancer stem/progenitor cells

Ming Luo et al. Cancer Res. .

Abstract

Focal adhesion kinase (FAK) has been implicated in the development of cancers, including those of the breast. Nevertheless, the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here, we show that _targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers, including aldehyde dehydrogenase, CD24, CD29, and CD61, we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-_targeted mice and impaired their self-renewal and migration in vitro. Finally, through transplantation in NOD-SCID mice, we found that cancer stem/progenitor cells isolated from FAK-_targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together, these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression.

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Figures

Figure 1
Figure 1. _targeted disruption of FAK in the mammary epithelium suppresses mammary tumor formation
(A) Kaplan-Meier analysis of mammary tumor development in the PosCNT (n=30), CreCNT (n=20) and _target (n=30) mice. _target vs PosCNT or CreCNT: P< 0.01 by the log-rank test. (B) Average number (±SD) of palpable mammary tumors per mouse per genotype at the indicated ages. _target versus PosCNT: P< 0.05 by the two-way ANOVA. (C) Representative mammary gland whole-mounts from 5-, 6- and 8-week-old PosCNT (a, c and e) and _target (b, d and f) mice. The arrow marks hyperplastic nodules. Scale bar= 5 mm. (D) Immunoblotting analysis of the lysates from primary tumors of 4 different (1-4) PosCNT and _target mice as well as mammary glands from the mice using antibodies against FAK, PyVT, and vinculin.
Figure 2
Figure 2. Analyses of protein expression, tumor growth and metastasis in _target mice
(A) Sections from the primary tumors of the PosCNT (a, c) and _target (b, d) mice were analyzed by immunohistochemistry using antibodies against FAK (a, b) or PyVT (c, d). The arrows mark blood vessels in panel b. Scale bars= 200 μm. (B) Lysates of 6 tumors in 3 different PosCNT (P1-P6) and _target (T1-T6) mice were prepared and analyzed by immunoblotting using antibodies against various proteins as indicated. (C) Mean cumulative mammary tumor volume (±SD) (upper panel) and weight (±SD) (low panel) for each genotype at indicated times after primary tumor appearance were plotted and analyzed. * P<0.05 and ** P<0.01 when _target mice is compared to either PosCNT or CreCNT mice. (D) Lung sections (4–6 sections per mouse) were prepared at 8 weeks after the primary mammary tumor onset, stained with hematoxylin and eosin (H&E) and the micrometastatic nodules were quantitated under a microscope. Upper panels are representative images with <1, 1–5, or >5 metastases per lung section, and lower panel shows percentages of mice of the indicated genotype with <1, 1–5, or >5 metastases per lung section. Scale bar = 2 mm. ** P<0.01 when _target mice is compared to either PosCNT or CreCNT mice.
Figure 3
Figure 3. Ablation of FAK depleted a subset of basal cells that co-express luminal and basal markers in the tumors of _target mice
(A) Sections from the primary tumor of CreCNT (a, c, e) and _target (b, d, f) mice were analyzed by immunohistochemistry using antibodies against CK8/18 (a, b) and CK5 (c-f). The arrows in panel c mark clusters of basal cells in the periphery of tumor nodules of CreCNT mice in early carcinoma stage, and arrowheads mark basal cells present in the tumor nodules. Panels e and f are from sections of tumor of CreCNT and _target mice, respectively, in late carcinoma stage. Scale bars= 200 μm. (B) Sections from the primary tumor of CreCNT (a,c,e,g) and _target (b,d,f,h) mice were analyzed by double labeling immunofluorescence using antibodies against CK8/18 and CK5. Arrows in panels g and h indicate bipotent cells with co-expression of both markers. Scale bars=200 μm.
Figure 4
Figure 4. Decreased MaCSC content in the primary tumors of _target mice
(A) Freshly isolated tumor cells from CreCNT (left) and _target (right) mice were depleted of CD45 and CD31 positive cells and labeled with CD24, CD29 and CD61 antibodies. The MaCSC population in each strain of mice was gated as Lin−CD24+CD29+CD61+ (upper right quad) under the same gating criteria. Results are representative of two separate experiments. In the second experiment, 58.32% and 28.08% Lin−CD24+ cells are CD29+CD61+ for CreCNT and _target tumor cells, respectively. (B) ALDEFLUOR assay of freshly isolated tumor cells of CreCNT (left panels) and _target (right panels) mice. The percentage of ALDH+ cells was determined under similar gating criteria (resided in gate R3). Results are representative of two separate experiments. In the second experiment, 23.37 % and 8.28 % tumor cells are ALDH+ for CreCNT and _target tumor cells, respectively (see Supplementary Fig. S2).
Figure 5
Figure 5. MaCSCs isolated from the primary tumors of the _target mice have reduced capacity for self-renewal and migration in vitro
(A) Freshly isolated tumor cells of a CreCNT mouse were sorted for ALDH+ and ALDH− cells, and analyzed for primary (P1) and secondary (P2) mammosphere formation under suspension culture conditions. Results are generated from eight separated incubations for each sample and are representative of two independent experiments. **P<0.001 in comparison to values for ALDH+ cells. (B) ALDH+ tumor cells of CreCNT and _target mice were analyzed for primary (P1) and secondary (P2) formation of mammospheres. Results are generated from six separated incubations for each sample and are representative of two independent experiments. Data for a separate experiment as well as images of mammospheres are shown in Supplementary Fig. S3C. **P<0.001 in comparison to values for cells isolated from CreCNT mice. Bar: 200 μm. (C) Unsorted, ALDH− and ALDH+ tumor cells of CreCNT mice were analyzed using Boyden chamber assay for their migratory capacity. Results are drawn from three independent experiments. (D) ALDH+ tumor cells of CreCNT and _target mice were analyzed in Boyden chamber assay to compare their migratory capacity. Results are drawn from three independent experiments. **P<0.001 in comparison to values for cells isolated from CreCNT mice. Representative images of migrated cells are shown in Supplementary Fig. S4.
Figure 6
Figure 6. MaCSCs isolated from the _target mice have reduced tumorigenicity and impaired maintenance in NOD-SCID mice following transplantation
(A) Tumor grown in a NOD-SCID mouse from 500 CreCNT ALDH+ tumor cells after 3 months of observation (arrow on the right side) whereas 500 _target ALDH+ tumor cells injected in the No.4 inguinal mammary fatpad (arrow on the left side) failed to generate tumor at the same time point. Bar=1 cm. (B) Tumor growth curves resulted from ALDH+ tumor cells of CreCNT and _target mice at different dilutions. (C) ALDEFLUOR assay of freshly isolated tumor cells from secondary tumors (at 1 month after their appearance) developed from 500 CreCNT (left panels) and 50,000 _target ALDH+ tumor cells (right panels). The percentage of ALDH+ cells for each tumor transplant was determined under similar gating criteria. Results are representative of two separate experiments. (D) Tumor cells dissociated from secondary tumors derived from 500 CreCNT (upper panel) or 50,000 _target ALDH+ tumor cells (lower panel) were depleted of CD45 and CD31 positive cells and labeled with CD24, CD29 and CD61 antibodies. The MaCSC population in each tumor transplant was gated as Lin−CD24+CD29+CD61+ under the same gating criteria. Results are representative of two separate experiments.
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