A requirement for Nedd9 in luminal progenitor cells prior to mammary tumorigenesis in MMTV-HER2/ErbB2 mice

Abstract

Overexpression of the NEDD9/HEF1/Cas-L scaffolding protein is frequent, and drives invasion and metastasis in breast, head and neck, colorectal, melanoma, lung and other types of cancer. We have examined the consequences of genetic ablation of Nedd9 in the MMTV-HER2/ERBB2/neu mouse mammary tumor model. Unexpectedly, we found that only a limited effect on metastasis in MMTV-neu;Nedd9(-/-) mice compared with MMTV-neu;Nedd9(+/+) mice, but instead a dramatic reduction in tumor incidence (18 versus 80%), and a significantly increased latency until tumor appearance. Orthotopic reinjection and tail-vein injection of cells arising from tumors, coupled with in vivo analysis, indicated tumors arising in MMTV-neu;Nedd9(-/-) mice had undergone mutational selection that overcame the initial requirement for Nedd9. To better understand the defects in early tumor growth, we compared mammary progenitor cell pools from MMTV-neu;Nedd9(-/-) versus MMTV-neu;Nedd9(+/+) mice. The MMTV-neu;Nedd9(-/-) genotype selectively reduced both the number and colony-forming potential of mammary luminal epithelial progenitor cells, while not affecting basal epithelial progenitors. MMTV-neu;Nedd9(-/-) mammospheres had striking defects in morphology and cell polarity. All of these defects were seen predominantly in the context of the HER2/neu oncogene, and were not associated with randomization of the plane of mitotic division, but rather with depressed expression the cell attachment protein FAK, accompanied by increased sensitivity to small molecule inhibitors of FAK and SRC. Surprisingly, in spite of these significant differences, only minimal changes were observed in the gene expression profile of Nedd9(-/-) mice, indicating critical Nedd9-dependent differences in cell growth properties were mediated via post-transcriptional regulation of cell signaling. Coupled with emerging data indicating a role for NEDD9 in progenitor cell populations during the morphogenesis of other tissues, these results indicate a functional requirement for NEDD9 in the growth of mammary cancer progenitor cells.

Figure 1. A Comparison of in vivo growth of MMTV-neu;Nedd9^+/+ versus MMTV-neu;Nedd9^−/− primary tumors and metastases

A. Kaplan-Meier curve indicates a significant increase in latency and decrease in incidence for tumor formation in MMTV-neu;Nedd9^−/− (n = 8 from 46 total) versus MMTV-neu;Nedd9^+/+ (n = 39 from 44 total) mice (P = 2.17e-09). B. Kaplan Meier curve indicates days intervening from initial detection of tumor to sacrifice for MMTV-neu;Nedd9^−/− versus MMTV-neu;Nedd9^+/+ mice (p = 0.01). C. Representative H&E-stained mammary tumors from MMTV-neu;Nedd9^+/+ and MMTV-neu;Nedd9^−/− tumors, at magnification 20X. D. The number of metastases per lung parenchyma cross sectional area (cm²) are not significantly different between MMTV-neu;Nedd9^+/+ and MMTV-neu;Nedd9^−/− mice with large primary tumors (p=0.51; mean and SEM are indicated as calculated using Wilcoxon rank sum tests).

Figure 2. Comparison of in vivo growth of MMTV-neu;Nedd9^+/+ versus MMTV-neu;Nedd9^−/− cell lines in orthotopic xenograft and tail vein injection, and analysis of genome stability

A. MMTV-neu;Nedd9^−/− (red) xenografts versus MMTV-neu;Nedd9^+/+ (blue) xenografts in SCID mice (p=0.1652). Data shown reflects each cell line according to genotype; solid lines represent average of 3 lines of each genotype. B. Quantification of lung metastases/cm², averaged from 5 mice/cell line (30 mice), 28 days after tail vein injection. MMTV-neu;Nedd9^−/− tumor cells were less metastatic to lung than MMTV-neu;Nedd9^+/+ cells (p=0.042). C. MMTV-neu;Nedd9^−/− (red) xenografts versus MMTV-neu;Nedd9^+/+ (blue) xenografts in MMTV-neu mice (p=0.2120). Data shown reflects each cell line according to genotype; solid lines represent average of 3 lines of each genotype. D. Western analysis of protein lysates of primary tumor tissue from MMTV-neu;Nedd9^+/+ versus MMTV-neu;Nedd9^−/− primary tumors (left) indicates no consistent differences in expression or phosphorylation (reflecting activation) for proteins shown. Representative Westerns blots from 3 experiments. +/−, mouse initially genotyped as Nedd9^−/−, but subsequently re-genotyped as Nedd9^+/− and excluded from all analyses. Western analysis with mammary cell lines derived from tumors indicated (right). E. Top, immunohistochemical staining for HER2/neu from representative H&E counterstained primary tumors. Bottom left, quantification on a scale of 1–4 for all tumors indicates no significant difference (p=0.33). Bottom right, western blot analysis of the primary mammary cell lines indicates similar HER2/neu levels, independent of Nedd9 genotype.

Figure 3. Comparative analysis of mammary progenitor cells in MMTV-neu;Nedd9^−/−, MMTV-neu;Nedd9^+/+, Nedd9^+/+, and Nedd9^−/− mice

A. Top, gating plots of FACS sorted mammary epithelial cells. Bottom, representative FACS plot showing relative proportion of CD24^high;CD49f^low cells harvested from at least 7 independent experiments. B. Top, quantification of percentage of luminal cell progenitor population within bulk mammary epithelial cells was reduced in Nedd9^−/− mice positive (p=0.018) and negative (p=0.015) for the MMTV-neu transgene. Bottom, similar analysis performed for the CD24^medCD49f^high basal cell progenitor pool indicates no significant difference based on Nedd9 genotype (p= 0.744 and 0.648, for neu positive and negative). * represents statistical significance by two-tailed t-test < 0.05.

Figure 4. Mammosphere growth and morphology in cells with MMTV-neu;Nedd9^−/−, MMTV-neu;Nedd9^+/+, Nedd9^+/+, and Nedd9^−/− genotypes

A. Average number of mammosphere colonies per 5,000 luminal progenitor cells seeded. Error bars indicate SEM. (p<0.0001). B. Quantification of average area (μm²) of each mammosphere colony indicates increase in overall area of mammospheres formed by MMTV-neu;Nedd9^−/− versus MMTV-neu;Nedd9^+/+ luminal progenitor cells (average 1623 μm² vs. 922 μm², p= 0.0022). C. Cell cycle compartmentalization of cells with genotypes indicated, determined by Guava analysis of cells recovered after growth in Matrigel. No significant differences were predicted by genotype. D. Frequency of condensed, pycnotic nuclei, reflecting apoptosis, in cells grown in Matrigel. No significant differences were associated with Nedd9 genotype; presence of MMTV-neu slightly increased rate of apoptosis. E. Immunofluorescence of luminal progenitor cell-derived colonies after 7–10 days in Matrigel. At least 3 independent experiments were performed. Green, phalloidin-488 indicates actin; blue, DAPI. Magnification, 40× oil; scale bar, 50 μm. F. Quantification of hollow, filled, or atypical colonies, from representative images shown in E. Error bars indicate SEM.

Figure 5. Defective FAK and SRC signaling in MMTV-neu;Nedd9^−/− versus MMTV-neu;Nedd9^+/+ mammospheres

A. Colonies visualized with antibodies to SRC or FAK, or with phalloidin, are shown as (a) image stack to represent entire mammosphere, (b) confocal section, and (c) pseudocolored image, where yellow represents the highest signal intensity and purple represents the lowest. Scale bar, 100 μm. B. Metamorph-based quantification of signal intensity in mammospheres in mammospheres, averaged from 8–11colonies per genotype. P values, *1. 0.0145, *2. 0.0659, *3. 0.0056, relative to Nedd9^+/+ control. C. Quantification of average area (μm²) of each mammosphere colony formed from luminal progenitor cells, following plating and maintenance in Matrigel with indicated doses of vehicle or PF-573228 for 10 days. Data are expressed in arbitrary scale. Significant P values (for drug-treated in comparison to vehicle treated for each genotype: 1) 0.770, 2) 0.9650, 3) 1.826^E-17, 4) 0.0002, 5) 0.0155, 6) 4.0336^E-17, 7) 0.6561, 8) 0.1916, 9) 2.225^E-14, 10) 0.6718, 11) 0.00084, and 12) 1.677^E-12. D. Analysis as in C, but instead following treatment with the SRC inhibitor dasatinib. Significant P-values include 1) 0.3395, and 2) 6.668^E-16. E. Left, representative images of mitotic spindles visualized with antibodies to a-tubulin in mammospheres grown for 6 days in Matrigel; insets represent expanded images. Right, average orientation of the spindle to the plane of the cell surface, reported as in (57). Bold lines represent average and dashed lines represent +/− standard deviation. No significant difference was observed between the MMTV-neu;Nedd9^−/−versus MMTV-neu;Nedd9^+/+ genotypes.