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SCIENCE CHINA Life Sciences, Volume 62 , Issue 2 : 225-234(2019) https://doi.org/10.1007/s11427-018-9336-6

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice

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  • ReceivedJun 30, 2018
  • AcceptedJul 23, 2018
  • PublishedNov 19, 2018

Abstract

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.


Funded by

grants from the Ministry of Science and Technology of China(2016YFC1302103,2015CB553906,2013CB910501)

the National Natural Science Foundation of China(81730071,81230051,81472734,31170711)

the Beijing Natural Science Foundation(7120002,7171005)

the 111 Project of the Ministry of Education

grants from Peking University(BMU20120314,BMU20130364)

and a Leading Academic Discipline Project of Beijing Education Bureau to H.Z. This work was also supported by a grant from the National Natural Science Foundation of China(81773199)


Acknowledgment

This work was supported by grants from the Ministry of Science and Technology of China (2016YFC1302103, 2015CB553906, and 2013CB910501), the National Natural Science Foundation of China (81730071, 81230051, 81472734, and 31170711), the Beijing Natural Science Foundation (7120002 and 7171005), the 111 Project of the Ministry of Education, grants from Peking University (BMU20120314 and BMU20130364), and a Leading Academic Discipline Project of Beijing Education Bureau to H.Z. This work was also supported by a grant from the National Natural Science Foundation of China (81773199) to J.Z.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Western blot analyses show the levels of Kindlin-2 in the WT and transgenic mice of the heart, liver, spleen, lung, thymus and brain tissues.

Figure S2 A. Whole mount staining of 2-week-old mammary glands. a. WT mammary gland. b. MMTV-Kindlin-2 mammary gland. B. Statistical analyses show the number of 2 weeks old mammary gland branches and end points. C. Statistical analyses exhibit the fat pad coverage percent of 2 weeks old mammary gland.

Table S1 Molecular and pathological type of mammary tumors from MMTV-Kindlin-2 mice

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Generation and characterization of MMTV-Kindlin-2 transgenic mice. A, The schematic diagram of the MMTV-Kindlin-2 construction. 2043 bp overall length human Kindlin-2 DNA fragment (the second white strip) was cloned and inserted into the MMTV specific promoter plasmid. B, Tail DNA was extracted and detected by PCR using Kindlin-2 specific primers, which make sure that Kindlin-2 has an insertion into the mouse genome or not. C, Western blot analysis was adopted to evaluate Kindlin-2 expression level in mammary glands tissues. The expression level of Kindlin-2 in transgenic group was significantly higher than WT group. D, Quantification and statistical analyses of Western blot analysis were performed and a significance difference was shown in the diagram. E, IHC was used to detect Kindlin-2 protein level in situ in virgin mouse mammary gland. Scale bar, 50 µm. Magnification of a and b are shown in c and d respectively. Black arrows indicate luminal cells and red arrows represent myoepithelial cells. F, Quantification and statistical analyses of IHC are shown in the chart.

  • Figure 2

    Kindlin-2 promotes mammary duct development and alveoli proliferation. A, Whole mount staining of 6-week-old virgin mammary glands. a, WT mammary gland; b, MMTV-Kindlin-2 mammary gland. Advanced development and more branches were observed in MMTV-Kindlin-2 group compared with WT group. B, Statistical analyses of 6-week-old MMTV-Kindlin-2 and wild type mammary gland with number of branches and end points. C, Statistical analyses with the filled fat pad percent of 6-week-old MMTV-Kindlin-2 and wild type mammary gland. D, Hematoxylin and eosin (HE) staining of 10-week-old virgin mammary glands. a, WT mammary glands; b, MMTV-Kindlin-2 mammary glands. Scale bar, 100 µm. Black arrows indicate mammary ducts. There are more branches of pipeline in MMTV-Kindlin-2 mammary glands than that in WT mammary glands. E, HE staining of mammary glands from 16.5 days pregnant mice. a, WT mammary glands; b, MMTV-Kindlin-2 mammary glands. Scale bar, 200 µm. Magnification of a and b are shown in c and d respectively. Scale bar, 50 µm. Black arrows represent alveolus in pregnant mammary gland. Red arrows represent secretion in the alveoli. Bigger acini and obvious secretions were observed in MMTV-Kindlin-2 mammary glands than that in WT mammary glands.

  • Figure 3

    Ectopic expression of Kindlin-2 upregulates CK5 and downregulates CK8 in MMTV-Kindlin-2 mammary glands. A, Immunohistochemistry detection for CK5 in mammary epithelium. a, CK5 in WT mammary glands; b, CK5 in MMTV-Kindlin-2 mammary glands. Magnifications of a and b are shown in the top left corner. Black arrows indicate luminal cells and red arrows represent myoepithelial cells. B, Quantification and statistical analyses of A is exhibited. C, Immunohistochemistry detection for CK8 in mammary epithelium. a, CK8 in WT mammary glands; b, CK8 in MMTV-Kindlin-2 mammary glands. Magnification of a and b are shown in the top left corner. Black arrows indicate luminal cells and red arrows represent myoepithelial cells. Scale bar, 50 µm. D, The histogram shows the quantification and statistical analyses of C.

  • Figure 4

    Kindlin-2 upregulates β-catenin in Kindlin-2 transgenic mammary glands. A, Ectopic expression of Kindlin-2 increases the level of β-catenin in MMTV-Kindlin-2 mammary glands. a, β-catenin expression in a longitudinal section of WT mammary glands; b, β-catenin expression in a longitudinal section of MMTV-Kindlin-2 mammary glands; c, β-catenin expression in a cross section of WT mammary glands; d, β-catenin expression in a cross section of MMTV-Kindlin-2 mammary glands. Scale bar, 20 µm. B, Elevated β-catenin level in MMTV-Kindlin-2 mammary gland is quantified (P=0.0492).

  • Figure 5

    Kindlin-2 promotes mammary tumorigenesis in transgenic mice. A, Tumor genesis in MMTV-Kindlin-2 transgenic mice. a, tumor bearing mice in MMTV-Kindlin-2 transgenic group; b, a tumor coming from MMTV-Kindlin-2 transgenic mice; c, H&E staining of the tumor from MMTV-Kindlin-2 transgenic mice. Scale bar, 100 µm. B, Kaplan-Meir analysis to show the tumor-free survival. C, Western blot detected expression of Kindlin-2, ERα, PR, Her2 and β-catenin in tumor tissues compared with para-tumor mammary gland tissues using specific antibodies. D, Quantification and statistical analyses of Kindlin-2, ERα, PR, Her2 and β-catenin protein in tumor tissues (T) and para-tumor mammary gland tissues (M) are used as control. E, Working model of Kindlin-2 promotes development of mammary gland in puberty and pregnant stages in mice and promotes breast tumor genesis in MMTV-Kindlin-2 transgenic mice.

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