Final Report Summary - CCING (Cadherin control of invasive growth in morphogenesis and cancer)
1. WORK PROGRESS AND ACHIEVEMENTS
This proposal aimed to determine how stage-dependent regulation of cadherins controls physiological invasive growth in normal epithelia and ill-fated invasive growth in epithelial cancer. As models, we focus on contrasting branching morphogenesis of kidney and mammary epithelia to neoplastic invasion of kidney and breast cancer cells.
A summary of progress towards objectives and details for each task
Model development: Potential models for normal physiological and neoplastic invasive growth of mammary epithelium were characterized for invasive growth and cadherin expression. For physiological invasive growth these included the human MCF10A line and the murine lines Eph4, HC11 and NMuMG lines. In reconstituted basement membrane (Matrigel) these lines show limited apical-basal polarization and are poorly or non-invasive. In collagen I-based matrices, only NMuMG, polarized to a limited extent, whereas all lines engaged in collective invasive growth, which was promoted by hepatocyte growth factor (HGF). EpH4 and NMuMG cells often invaded with blunted buds with no leader or tip cell, similar as in normal mammary development in vitro. The MCF10A, Eph4 and HC11 lines suffered from variable levels of invasive growth within the same culture which confounded temporal quantitative analyses. Efforts to lower heterogeneity by varying culturing conditions or by subcloning were not sufficiently effective, indicating that phenotypic heterogeneity is cell-intrinsic, consistent with other published models we screened and extensively reviewed in Sem Cell Dev Biol [1]. In contrast, spontaneous invasive growth of NMuMG cells in collagen exhibited a robust and homogenous response. We thus chose NMuMG cell line as primary model as it allowed for reliable probing of physiological invasive growth and retained MCF10A, Eph4 and HC11 cells as models to validate results obtained in the NMuMG model.
As tumorigenic counterpart we established 3D culture of a mouse mammary tumor progression model, consisting of the 67NR, 168FARN, 4T07 and 4T1 cell lines. These cells were derived from a single mammary tumor but differ in local invasion in vivo, including formation of primary tumors without dissemination (67NR), dissemination into lymph nodes only (168FARN), formation of lung micrometastasis (4TO7), and completion of all steps up to distant macrometastasis (4T1) [2]. We found that in 3D collagen culture, 67NR cells did not invade, 168FARN collectively invaded at low levels, and 4T07 and 4T1 showed extensive invasion as single cells (4T07) or predominantly as collective strands (4T1). This may indicate that metastatic progression involves both individual-cell and collective dissemination modes at different stages of the cascade.
Due to its superior characteristics in cell polarization and the capability to undergo staged tubulogenesis upon treatment HGF [1], we retained the MDCK line as model for normal invasive growth, with the murine mIMCD3 line [1] as additional model for data validation.
Molecular characterization. The mammary models revealed unique cadherin expression patterns for each line. Key observations were that NMuMG and 4T1 cells were the only lines expressing E-cadherin, and that168FARN and 67NR predominantly express P-cadherin. In contrast to 67NR, expression of a- and b-catenin was undetectable by western blot in 168FARN, indicating that these cells do not form P-cadherin-based adherens junctions. Similarly, 4T07 likely do not form adherens junctions as no a- and b-catenin, or any cadherin thus far examined (E-, N-, P-cadherin or cadherin-6,or -11) was detected. Similar to our previous findings in MDCK [3], invasive growth of NMuMG or 4T1 was not associated with global downregulation of E-cadherin when analyzed in immunofluorescence-based studies. However, a fraction of cellular E-cadherin was cleaved in cells grown in 3D matrices, but not on tissue culture plastic. The main cleavage products are a 80 kD soluble and a 33 kD cytosolic fragment, which were previously implicated by others in promoting mammary tumorigenesis. Our preliminary data indicate an involvement of calpain, but not matrix metalloproteases. To investigate local induction of EMT, and associated inhibition of E-cadherin expression, NMuMG and EpH4 cells were transfected with the EGFP-linked promoter reporter construct for the SNAI2 gene, which codes for the transcriptional repressor Slug. Preliminary microscopy data indicate that whereas invasive growth is associated with local induction of Slug, this did not directly correlate with downregulation of E-cadherin, something that was previously also observed in the MDCK model [4]. In ongoing work, the expression plasticity of cell-cell junction proteins is evaluated for its impact on invasion mode and impact on cell growth in 3D models.
A summary of the progress of the researcher training activities/transfer of knowledge activities/integration activities (as it applies for the MC action)
Researcher training activities of the researcher:
Dr. Zegers successfully established cancer models and molecular reporter assays and thus widened her strategic portfolio. As ongoing training activity, to validate molecular targets identified in this project, she will apply intravital microscopy in small animals. Consequently,, Dr. Zegers successfully completed the course on laboratory animal science (3 ECTS), which comprises the requirements cited in article 9 of Experiments on Animals Act and the European FELASA category C, and a legal recognition to design animal experiments in the Netherlands, and is currently being trained in applying imaging window models.
Transfer of knowledge by the researcher:
During the period covered by this project, Dr. Zegers directly supervised and trained several students in the theory and practice of epithelial cell biology, invasive growth and cancer progression. This included 6-month internships of two M.Sc students of the international master program Molecular Mechanisms of Disease (MMD), a highly selective master program of which over 80% of the student body comes from EU member states. Guest lectureships for the MMD program provided additional training in molecular mechanisms of cell migration. Additional students trained included two M.Sc. students in biomedical science (Radboud University, Nijmegen) and one bachelor student Life Science (HAN University of Applied Sciences, Nijmegen).
The synergism of the combined expertise of Drs. Friedl and Zegers facilitated by this IIF-CCinG project has directly led to the successful application for a competitive Institute-funded PhD position on regulation of adhesion molecules by the tumor-microenvironment. This project addresses how p120 is regulated by growth factors and physicochemical characteristics of the extracellular matrix and thus is complementary to the CCinG project. This PhD student is jointly supervised by Drs. Friedl and Zegers, thereby securing long-term implementation of the expertise of Dr. Zegers into Dr. Friedl’s team. Finally, the combined expertise of Drs. Friedl and Zegers has led to their co-authorship on two publications, one of which appeared in the high impact journal Nature Cell Biology [5, 6].
Highlight clearly significant results
Significant results towards objectives include : The establishment and of relevant models for physiological and ill-fated invasive growth of the mammary gland; the morphological characterization of invasion strategy and, partially, the associated molecular characterization of cadherin profile; the identification of biologically relevant E-cadherin cleavage products in an invasive mammary tumor line with potential implications for neoplastic invasive growth.
Significant results of knowledge transfer include: Practical and theoretical training of several BSc and MSc level students; acquisition of a project-related PhD position, funded by the Radboud Institute for Molecular Life Sciences.
If applicable, explain the reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning
Not applicable.
If applicable, explain the reasons for failing to achieve critical objectives and/or not being on schedule and explain the impact on other tasks as well as on available resources and planning (the explanations should be coherent with the declaration by the project coordinator)
Not applicable.
2. ADDITIONAL INFORMATION
Not applicable
3. PROJECT MANAGEMENT
The work is proceeding as planned, and no deviations of project management have occurred. We only wish to report a name change of the Institute that comprises the host laboratory of Cell Biology, to Radboud Institute of Molecular Life Sciences (previously known as the Nijmegen Centre for Molecular Life Sciences). This name change has no consequences for the project.
REFERENCES
References comprising output from this project are indicated with asterisk.
*[1] Zegers MM. 3D in vitro cell culture models of tube formation. Semin Cell Dev Biol 2014;10.1016/j.semcdb.2014.02.016.
[2] Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 1992;52:1399-405.
[3] Jia L, Liu F, Hansen SH, Ter Beest MB, Zegers MM. Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation. Mol Biol Cell 2011;22:2031-41.
[4] Leroy P, Mostov KE. Slug is required for cell survival during partial epithelial-mesenchymal transition of HGF-induced tubulogenesis. Mol Biol Cell 2007;18:1943-52.
*[5] Friedl P, Wolf K, Zegers MM. Rho-directed forces in collective migration. Nat Cell Biol 2014;16:208-10.
*[6] Zegers MM, Friedl P. Rho GTPases in collective cell migration. Small GTPases 2014;5:e28997.
This proposal aimed to determine how stage-dependent regulation of cadherins controls physiological invasive growth in normal epithelia and ill-fated invasive growth in epithelial cancer. As models, we focus on contrasting branching morphogenesis of kidney and mammary epithelia to neoplastic invasion of kidney and breast cancer cells.
A summary of progress towards objectives and details for each task
Model development: Potential models for normal physiological and neoplastic invasive growth of mammary epithelium were characterized for invasive growth and cadherin expression. For physiological invasive growth these included the human MCF10A line and the murine lines Eph4, HC11 and NMuMG lines. In reconstituted basement membrane (Matrigel) these lines show limited apical-basal polarization and are poorly or non-invasive. In collagen I-based matrices, only NMuMG, polarized to a limited extent, whereas all lines engaged in collective invasive growth, which was promoted by hepatocyte growth factor (HGF). EpH4 and NMuMG cells often invaded with blunted buds with no leader or tip cell, similar as in normal mammary development in vitro. The MCF10A, Eph4 and HC11 lines suffered from variable levels of invasive growth within the same culture which confounded temporal quantitative analyses. Efforts to lower heterogeneity by varying culturing conditions or by subcloning were not sufficiently effective, indicating that phenotypic heterogeneity is cell-intrinsic, consistent with other published models we screened and extensively reviewed in Sem Cell Dev Biol [1]. In contrast, spontaneous invasive growth of NMuMG cells in collagen exhibited a robust and homogenous response. We thus chose NMuMG cell line as primary model as it allowed for reliable probing of physiological invasive growth and retained MCF10A, Eph4 and HC11 cells as models to validate results obtained in the NMuMG model.
As tumorigenic counterpart we established 3D culture of a mouse mammary tumor progression model, consisting of the 67NR, 168FARN, 4T07 and 4T1 cell lines. These cells were derived from a single mammary tumor but differ in local invasion in vivo, including formation of primary tumors without dissemination (67NR), dissemination into lymph nodes only (168FARN), formation of lung micrometastasis (4TO7), and completion of all steps up to distant macrometastasis (4T1) [2]. We found that in 3D collagen culture, 67NR cells did not invade, 168FARN collectively invaded at low levels, and 4T07 and 4T1 showed extensive invasion as single cells (4T07) or predominantly as collective strands (4T1). This may indicate that metastatic progression involves both individual-cell and collective dissemination modes at different stages of the cascade.
Due to its superior characteristics in cell polarization and the capability to undergo staged tubulogenesis upon treatment HGF [1], we retained the MDCK line as model for normal invasive growth, with the murine mIMCD3 line [1] as additional model for data validation.
Molecular characterization. The mammary models revealed unique cadherin expression patterns for each line. Key observations were that NMuMG and 4T1 cells were the only lines expressing E-cadherin, and that168FARN and 67NR predominantly express P-cadherin. In contrast to 67NR, expression of a- and b-catenin was undetectable by western blot in 168FARN, indicating that these cells do not form P-cadherin-based adherens junctions. Similarly, 4T07 likely do not form adherens junctions as no a- and b-catenin, or any cadherin thus far examined (E-, N-, P-cadherin or cadherin-6,or -11) was detected. Similar to our previous findings in MDCK [3], invasive growth of NMuMG or 4T1 was not associated with global downregulation of E-cadherin when analyzed in immunofluorescence-based studies. However, a fraction of cellular E-cadherin was cleaved in cells grown in 3D matrices, but not on tissue culture plastic. The main cleavage products are a 80 kD soluble and a 33 kD cytosolic fragment, which were previously implicated by others in promoting mammary tumorigenesis. Our preliminary data indicate an involvement of calpain, but not matrix metalloproteases. To investigate local induction of EMT, and associated inhibition of E-cadherin expression, NMuMG and EpH4 cells were transfected with the EGFP-linked promoter reporter construct for the SNAI2 gene, which codes for the transcriptional repressor Slug. Preliminary microscopy data indicate that whereas invasive growth is associated with local induction of Slug, this did not directly correlate with downregulation of E-cadherin, something that was previously also observed in the MDCK model [4]. In ongoing work, the expression plasticity of cell-cell junction proteins is evaluated for its impact on invasion mode and impact on cell growth in 3D models.
A summary of the progress of the researcher training activities/transfer of knowledge activities/integration activities (as it applies for the MC action)
Researcher training activities of the researcher:
Dr. Zegers successfully established cancer models and molecular reporter assays and thus widened her strategic portfolio. As ongoing training activity, to validate molecular targets identified in this project, she will apply intravital microscopy in small animals. Consequently,, Dr. Zegers successfully completed the course on laboratory animal science (3 ECTS), which comprises the requirements cited in article 9 of Experiments on Animals Act and the European FELASA category C, and a legal recognition to design animal experiments in the Netherlands, and is currently being trained in applying imaging window models.
Transfer of knowledge by the researcher:
During the period covered by this project, Dr. Zegers directly supervised and trained several students in the theory and practice of epithelial cell biology, invasive growth and cancer progression. This included 6-month internships of two M.Sc students of the international master program Molecular Mechanisms of Disease (MMD), a highly selective master program of which over 80% of the student body comes from EU member states. Guest lectureships for the MMD program provided additional training in molecular mechanisms of cell migration. Additional students trained included two M.Sc. students in biomedical science (Radboud University, Nijmegen) and one bachelor student Life Science (HAN University of Applied Sciences, Nijmegen).
The synergism of the combined expertise of Drs. Friedl and Zegers facilitated by this IIF-CCinG project has directly led to the successful application for a competitive Institute-funded PhD position on regulation of adhesion molecules by the tumor-microenvironment. This project addresses how p120 is regulated by growth factors and physicochemical characteristics of the extracellular matrix and thus is complementary to the CCinG project. This PhD student is jointly supervised by Drs. Friedl and Zegers, thereby securing long-term implementation of the expertise of Dr. Zegers into Dr. Friedl’s team. Finally, the combined expertise of Drs. Friedl and Zegers has led to their co-authorship on two publications, one of which appeared in the high impact journal Nature Cell Biology [5, 6].
Highlight clearly significant results
Significant results towards objectives include : The establishment and of relevant models for physiological and ill-fated invasive growth of the mammary gland; the morphological characterization of invasion strategy and, partially, the associated molecular characterization of cadherin profile; the identification of biologically relevant E-cadherin cleavage products in an invasive mammary tumor line with potential implications for neoplastic invasive growth.
Significant results of knowledge transfer include: Practical and theoretical training of several BSc and MSc level students; acquisition of a project-related PhD position, funded by the Radboud Institute for Molecular Life Sciences.
If applicable, explain the reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning
Not applicable.
If applicable, explain the reasons for failing to achieve critical objectives and/or not being on schedule and explain the impact on other tasks as well as on available resources and planning (the explanations should be coherent with the declaration by the project coordinator)
Not applicable.
2. ADDITIONAL INFORMATION
Not applicable
3. PROJECT MANAGEMENT
The work is proceeding as planned, and no deviations of project management have occurred. We only wish to report a name change of the Institute that comprises the host laboratory of Cell Biology, to Radboud Institute of Molecular Life Sciences (previously known as the Nijmegen Centre for Molecular Life Sciences). This name change has no consequences for the project.
REFERENCES
References comprising output from this project are indicated with asterisk.
*[1] Zegers MM. 3D in vitro cell culture models of tube formation. Semin Cell Dev Biol 2014;10.1016/j.semcdb.2014.02.016.
[2] Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 1992;52:1399-405.
[3] Jia L, Liu F, Hansen SH, Ter Beest MB, Zegers MM. Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation. Mol Biol Cell 2011;22:2031-41.
[4] Leroy P, Mostov KE. Slug is required for cell survival during partial epithelial-mesenchymal transition of HGF-induced tubulogenesis. Mol Biol Cell 2007;18:1943-52.
*[5] Friedl P, Wolf K, Zegers MM. Rho-directed forces in collective migration. Nat Cell Biol 2014;16:208-10.
*[6] Zegers MM, Friedl P. Rho GTPases in collective cell migration. Small GTPases 2014;5:e28997.