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, 21 (1), 63

Absence of Integrin α3β1 Promotes the Progression of HER2-driven Breast Cancer in Vivo

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Absence of Integrin α3β1 Promotes the Progression of HER2-driven Breast Cancer in Vivo

Veronika Ramovs et al. Breast Cancer Res.

Abstract

Background: HER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3β1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3β1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis.

Methods: To investigate the role of α3β1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3β1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3β1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence.

Results: Tumor onset in mice was independent of the presence of α3β1. In contrast, the depletion of α3β1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3β1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow.

Conclusion: Downregulation of α3β1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3β1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3β1 in breast cancer.

Keywords: Adhesion; Breast cancer; Collagen; Extracellular matrix; HER2; Integrin α3β1; Interstitial fluid flow; MMTV-cNeu; Metastasis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Integrin α3β1 is not needed for the onset of HER2-driven mammary tumorigenesis. a Kaplan-Meier plots of tumor-free survival in Itga3 KO and WT mice show that first palpable tumors could be detected at comparable age in both genotypes. b The number of tumors, measured macroscopically over time, was similar between Itga3 KO and WT mice (n = 17). c The western blot of four randomly selected, representative tumors isolated from Itga3 KO and WT mice shows a clear deletion of α3 in Itga3 KO mice. d Representative images of immunohistochemical staining for HER2/Neu in Itga3 KO and WT tumors show its strong expression, which was observed in all the analyzed tumors. e Representative images of immunofluorescent staining show the absence of basal markers β4, keratin 5, and laminin-332, which is also the main ligand for integrin α3β1. E-cadherin, β-catenin, Plet1, collagen I, and keratin 18 were strongly expressed in all tumors. No difference was observed between Itga3 KO and WT mice. Scale bar, 200 μm
Fig. 2
Fig. 2
The absence of integrin α3 promotes HER2-dependent tumor growth and vascularization. a Survival Kaplan-Meier plots comparing Itga3 KO and WT mice. The median age when half of the mice per group needed to be sacrificed was 180 days for Itga3 KO mice and a month later (212 days) for Itga3 WT mice (n = 17). b Itga3 KO mice developed significantly bigger tumors from the age of 21 weeks on (n = 17; unpaired t test, *P < 0.05, **P < 0.005). c No difference in the number of proliferating Ki67-positive tumor cells was observed between Itga3 KO and WT mice. Left—Ki67-positive cells were counted in five fields of randomly selected tumors from eight mice per genotype (unpaired t test, P = 0.7168). Right—representative tumor images of immunohistochemical staining for Ki67. d, e Plots showing the fitted linear regression of average tumor sizes during d first and e last 3 weeks of tumor growth per mouse. The slopes of trend lines are significantly different during the first weeks of tumorigenesis, showing an increased growth rate in Itga3 KO mice. Such difference is not observed during the last 3 weeks of tumor growth before mice were sacrificed (n = 17, unpaired t test, *P < 0.05). f Vascularity of tumors, quantified as integrated density of CD31 stained samples (left), was increased in Itga3 KO mice. Five fields of randomly selected tumors from nine WT and ten KO mice were analyzed (unpaired t test, P = 0.0176). Right—representative tumor images of immunohistochemical staining for CD31
Fig. 3
Fig. 3
The absence of integrin α3 increases the invasiveness of HER2-driven tumors. a Significantly more Itga3 KO compared to WT mice developed lung metastasis (chi-square (Fisher’s exact) test). b The number of metastasis, counted in H&E-stained sections of the lungs, was significantly increased in Itga3 KO mice (unpaired t test, P = 0.0119). c Metastases were of similar sizes in Itga3 KO and WT mice (unpaired t test, P = 0.2222). d Itga3 KO compared to WT mice had a significantly larger total metastatic burden, i.e., total metastatic area in the lungs (chi-square test). e Representative images of invasive and blood-borne metastasis in the lungs of Itga3 KO and WT mice, stained for HER2/Neu. f The percentage of blood-borne and invasive out of total pulmonary metastases shows a small increase in the number of invasive metastases in Itga3 KO mice. g The percentage of invasive metastasis-bearing mice per genotype out of the total number of metastases-bearing mice. Almost twice as many Itga3 KO as WT mice displayed invasive metastases
Fig. 4
Fig. 4
Decreased ITGA3 expression in HER2+, compared to triple-negative human carcinoma cells. a Flow cytometry histograms of signal intensity for α3 (left) and HER2 (right) staining of six human mammary carcinoma cell lines show lower surface expression of α3 in HER2+ cells. 50 × 103 cells were analyzed per experiment; representative graphs of three independent experiments. b Scatter plot of ITGA3 and ERBB2 expression for HER2+ and triple-negative-enriched breast cancer panel [22] shows a negative correlation between ITGA3 and ERBB2 expression (Spearman’s rho − 0.46, P = 0.01, n = 30). c Scatter plot of ITGA3 gene copy number estimates against ITGA3 expression for HER2+ and triple-negative-enriched breast cancer panel [22]. Despite ITGA3 amplification in several HER2+ cell lines, their expression of ITGA3 remains relatively low
Fig. 5
Fig. 5
α3 reduction causes increased invasiveness of HER2+, but not triple-negative human carcinoma cells. a Flow cytometry histograms of signal intensity of α3 show successful α3 deletion in AU565, SKBR3, BT474, and MDA-MB-231cells. bd Invasion assays through gel-coated (Matrigel-collagen I or Matrigel only) membrane. Bottom—representative images of the part of the membrane, showing DAPI-stained nuclei of invading cells. Top—analysis of the experiments, performed in duplicate and repeated three times. (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005) b The reduction of α3 increases the invasiveness of HER2+ carcinoma cells through the mixture of collagen I and Matrigel under interstitial fluid flow conditions. c Under static conditions, HER2+ cells show strongly reduced and α3-independent invasion. Contrary, the invasion of triple-negative MDA-MB-231 is α3-dependent. d The absence of collagen I causes an increased invasion of α3WT HER2+ carcinoma cells, resulting in similar levels of invasion between α3KO and WT cells
Fig. 6
Fig. 6
α3-mediated changes in collagen adhesion and FA formation. a Short-term adhesion assays of HER− MDA-MB-231 and HER2+ BT474, AU565, and SKBR3 on laminin-rich matrix and collagen I. Experiments were performed in triplicate and repeated three times (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0001). b α3KO SKBR3 show significantly decreased cell area, c reduced size of focal adhesions, and d reduced total adhesion area per cell after 30 min of adhesion to collagen I. Experiments were performed three times with 30 cells analyzed per experiment (total n = 90, unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005). e Representative images of SKBR3 ITGA3 KO and WT cell, used for quantifications (bd), stained for actin and vinculin (scale bar, 10 μm). f Representative images of SKBR3 ITGA3 KO and WT cells, allowed to adhere to collagen I-coated coverslips for 30 min and stained for actin and integrins α3, α2, or β1. ITGA3 KO cells show reduced clustering of integrins in adhesion complexes. Note that α3 and α2 signals were enhanced by biotin-conjugated secondary antibody, resulting in unspecific biotin staining in the center of the cell (Additional file 4: Figure S4b) (scale bar, 10 μm). g Invasion assays through the mixture of collagen I and Matrigel-coated membrane. The reduction of α3 increases invasiveness of HER2+ carcinoma cells under interstitial fluid flow conditions, which cannot be recapitulated with blocking adhesion of α3 to laminin by addition of function-blocking J143 antibody (A3-X8: control non-blocking antibody). Left—analysis of the experiments, performed in duplicate and repeated three times (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005). Right—representative images of the part of the membrane, showing DAPI-stained nuclei of invading cells

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