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. 2010;12(3):R38.
doi: 10.1186/bcr2592. Epub 2010 Jun 21.

Cooperative signaling between Wnt1 and integrin-linked kinase induces accelerated breast tumor development

Arusha Oloumi  1 , Mykola Maidan, Frances E Lock, Howard Tearle, Steven McKinney, William J Muller, Samuel A J R Aparicio, Shoukat Dedhar
Affiliations

Cooperative signaling between Wnt1 and integrin-linked kinase induces accelerated breast tumor development

Arusha Oloumi et al. Breast Cancer Res. 2010.

Abstract

Introduction: Breast cancer is genetically and clinically a heterogeneous disease. However, the exact contribution of different cell types and oncogenic mutations to this heterogeneity are not well understood. Recently, we discovered an interaction between Wnt and integrin-linked kinase (ILK) within the signaling cascade that regulates cell growth and survival. Interestingly, mammary-specific expression of either one of these proteins has been shown to promote mammary tumorigenesis. In light of our recent findings and to investigate the potential interaction between Wnt and ILK proteins during mammary tumor formation and progression, we established a transgenic mouse model that expresses both Wnt and ILK in mammary epithelial cells.

Methods: A novel transgenic mouse model with mammary-specific expression of both Wnt1 and ILK was generated by crossing the two previously characterized mouse models, MMTV-Wnt1 and MMTV-ILK. The resulting MMTV-Wnt/ILK mice were closely monitored for tumor development and growth, as well as for the tumor onset. The molecular phenotypes of both tumors and premalignant mammary glands were investigated by using biochemical and global gene-expression analysis approaches.

Results: A significant acceleration in mammary tumor incidence and growth was observed in the MMTV-Wnt/ILK mice. Pre-neoplastic mammary glands also display lobuloalveolar hyperplasia and an increase in ductal epithelium proliferation. Apart from elevated expression of Wnt/ILK targets, such as beta-catenin and cyclin D1, gene-expression profiling identified the surprising activation of the FOXA1 transcription factor. Upregulation of FOXA1, which is also known as the molecular marker of differentiated mammary luminal cells, was consistent with the expansion of the enriched luminal progenitor population or CD29loCD24hiCD61+ cells in MMTV-Wnt/ILK tumors.

Conclusions: These results show cooperation between Wnt1 and ILK transgenes during mammary carcinogenesis, leading to changes in a transcriptional network, which could dictate a specific breast cancer phenotype with enhanced growth dynamics. The MMTV-Wnt/ILK can be used as a model to identify further the genes downstream of the estrogen receptor-beta/FOXA1 and to investigate the mechanisms targeting the expansion of the luminal progenitor cells leading to hyperplasia and tumorigenesis.

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Figures

Figure 1
Figure 1
Overexpression of the human ILK gene. (a) Mouse MMTV-Wnt1 and human MMTV-ILK transgene constructs (b). Quantitative RT-PCR performed on randomly selected tumors from each MMTV-Wnt1 and MMTV-Wnt/ILK group (n = 8) showing a dramatic overexpression of the human ILK gene in the double-transgenic samples. The bar graphs show the relative amount of human ILK overexpression in the individual MMTV-Wnt/ILK and MMTV-Wnt1 tumors normalized for β-actin expression. The statistical analysis with 95% confidence interval of (+754.18; -256.46) shows an average value of about 400-fold of overexpression in human ILK in MMTV-Wnt/ILK with an adjusted P value of 3.1E-16 determined by Benjamini-Hochberg test.
Figure 2
Figure 2
Mammary tumor formation and tumor growth in MMTV-Wnt/ILK mice. (a) Kaplan-Meier analysis of the percentage of tumor-free mice over time; P < 0.001. (b) Linear representation of growth rate from three randomly selected tumors from each transgenic group shows an accelerated rate of growth in MMTV-Wnt/ILK mice. (c) Tumor-doubling time was calculated based on the exponential curve fit of the tumor-volume measurements against time. The results show the mean doubling time for n = 7 tumors collected in each group, which supports the proliferative advantage of the tumors arising in the MMTV-Wnt/ILK transgenic group. The error bars represent the standard error of the mean (SEM), and the **P value was calculated by using a paired two-sample t test with a 95% confidence interval.
Figure 3
Figure 3
Pre-neoplastic mammary gland analysis. (a) Whole-mount ductal outgrowths from 10- to 12-week-old FVB, MMTV-Wnt1, and MMTV-ILK mice and 8-week-old MMTV-Wnt/ILK mice show a marked enhancement in the ductal network of MMTV-Wnt/ILK pre-neoplastic mammary glands at an earlier age. Images were taken by using a dissecting microscope at 4× magnification. Black arrow shows an area of dense alveoli. (b) Sections from inguinal mammary glands of 8- to 9-week-old mice were stained with H&E and PCNA. Mammary gland taken from an FVB mouse shows a normal glandular epithelial (a,b) and minimal PCNA staining (g-h), whereas sections from MMTV-Wnt show more hyperplasia (c,d) and more PCNA staining (i,j). Section of mammary glands from MMTV-Wnt/ILK mice shows an even greater epithelial hyperplasia, multilayered and disorganized ductal epithelium (e,f), as well as a marked increase in PCNA staining (k,l). The pictures are representative of at least five different stainings. Scale bars, 50 μm. (c) Ki67 was used as an additional marker to confirm the higher proliferation status of the MMTV-Wnt/ILK pre-neoplastic mammary glands. Scale bars, 100 μm. (d) Ki67-positive staining is quantified based on the number of the mammary lumina containing Ki67-positive cells. At least 50 mammary lumina were counted in each condition in randomly selected fields of view. Data represent mean ± SD. *P < 0.04 was calculated by using a paired two-sample t test.
Figure 4
Figure 4
Biochemical analysis, densitometry results, and immunochemistry. (a) The biochemical analysis of the tumors from each group demonstrates upregulation of certain molecular markers in MMTV-Wnt/ILK tumors, which could potentially lead to the more-aggressive growth phenotype observed in these tumors. The lysates from two different FVB mammary glands were used as controls. The blots were generated at the same time, by using the same apparatus, and under identical exposure times and conditions to ensure comparability. (b) The graph for the Western-blot analysis illustrates the densitometry results of the blots. Results represent the mean of densitometry data from 10 independent tumors normalized for β-actin expression. The error bars represent the standard error of the mean (SEM). *P ≤ 0.05; **P < 0.005; calculated by using an ANCOVA analysis. (c) β-catenin immunohistochemistry shows a significant increase in nuclear localization in excised tumors from MMTV-Wnt/ILK mice, suggesting a more-active β-catenin-mediated transcriptional machinery. Magnification: (a,b) 40×; (c,d) 63×; n = 4. β-catenin staining of the normal FVB mammary gland is included as a control (e,f). Scale bars, 25 μm. (d) Positive nuclear β-catenin staining was determined by counting the cells that showed overlapping hematoxylin and β-catenin staining. The cells were counted in five equal randomly chosen fields of view at 40× magnification in four individual tumors. Results are reported as the mean of the ratio of the total number of the nuclear-positive cells to the total cell count. The error bars represent the standard error of the mean (SEM). **P < 1.1 × 10-7 was calculated by using a paired two-sample t test.
Figure 5
Figure 5
Q-RT-PCR validation of the microarray data shows differential expression of 17 of 26 genes tested (at least three and up to eight samples). Results were exponentiated to obtain natural-scale fold-change estimates (that is, 2-ΔΔCt) and 95% confidence intervals, shown as bar heights and whiskers, respectively. The genes that showed a fold change of ≥ 30% with P ≤ 0.05 are presented as validated genes.
Figure 6
Figure 6
Immunohistochemical staining and PCR analysis. (a) Immunohistochemical staining of tumors from MMTV-Wnt/ILK mice exhibit strikingly enhanced expression of FOXA1 and ER-α. Magnifications: (a,d) 40×; (b,c, and e,f) 63×. Scale bars, 50 μm. (b) Q-RT-PCR analysis shows a 2.4-fold increase in ER-α transcripts levels in MMTV-Wnt/ILK tumors. Results are shown as fold change (FC) of ER-α expression in MMTV-Wnt/ILK over MMTV-Wnt1 tumors.
Figure 7
Figure 7
Flow-cytometry analysis and relative proportions of cell types. (a) Flow-cytometry analysis showing the distribution of Lin-CD29lo for the expression of the surface markers CD24 and CD61. Mammary glands from 10-week-old FVB mice were included as a control for the expression of these cell-surface markers and analysis by FACS. The increase in CD24hiCD61+ luminal progenitor cells is evident in the tumor cells of the MMTV-Wnt/ILK transgenic model compared with MMTV-Wnt (n = 3 per group). (b) Bar chart depicting the relative proportion of the CD61+ luminal progenitor cells to the total luminal population (CD29loCD24hi) in the two tumor types. Error bars indicate mean ± SD. **P < 0.002 was calculated by using a paired two-sample t test. (c) The purity of the cell populations (myoepithelial and luminal) in the sorted fractions is shown by the lineage-specific markers, K18 and K14, in the cultured tumor cells of both transgenic models. Scale bars, 25 μm.
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References

    1. Hanahan D, Wagner EF, Palmiter RD. The origins of oncomice: a history of the first transgenic mice genetically engineered to develop cancer. Genes Dev. 2007;21:2258–2270. doi: 10.1101/gad.1583307. - DOI - PubMed
    1. Veeck J, Niederacher D, An H, Klopocki E, Wiesmann F, Betz B, Galm O, Camara O, Durst M, Kristiansen G, Huszka C, Knuchel R, Dahl E. Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis. Oncogene. 2006;25:3479–3488. doi: 10.1038/sj.onc.1209386. - DOI - PubMed
    1. Sims AH, Howell A, Howell SJ, Clarke RB. Origins of breast cancer subtypes and therapeutic implications. Nat Clin Pract Oncol. 2007;4:516–525. doi: 10.1038/ncponc0908. - DOI - PubMed
    1. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–480. doi: 10.1016/j.cell.2006.10.018. - DOI - PubMed
    1. Cowin P. Unraveling the cytoplasmic interactions of the cadherin superfamily. Proc Natl Acad Sci USA. 1994;91:10759–10761. doi: 10.1073/pnas.91.23.10759. - DOI - PMC - PubMed

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