Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis

Abstract

Background: In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.

Methods: Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.

Results: We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.

Conclusions: We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Keywords: Collective cell migration; Metastasis; Non-canonical Wnt; Planar cell polarity.

Fig. 1

Wnt/PCP signaling mediates breast cancer cell migration. a Relative cell migration quantification of BT549, MDA-MB-231,MDA-MB-468, SkBr3, NDL, MCF7, and T47D cells stably expressing Control, shVangl2-1, or shVangl2-2 (BT549 n = 3, p = 4.81E-05 and p = 4.47E-05, MDA-MB-231 n = 4, p = 0.0001 and p = 2.73E-05, MDA-MB-468 n = 3, p = 0.0078 and p = 0.0146, SkBr3 n = 3, p = 0.003 and p = 0.0057, NDL n = 3, p = 0.0250 and p = 0.0063, MCF7 n = 3, p = 0.0180 and p = 0004, T47D n = 3, p = 2.13E-05 and p = 1.14E-06). b Representative images of migrating NDL cells stimulated with Vector- or Wnt5a- conditioned media at 0 and 12 h. Scale bar = 200 µm. c Relative cell migration quantification of MDA-MB-231, MDA-MB-468, SkBr3, NDL, and MCF7 cells stimulated with Vector- or Wnt5a-conditioned media (MDA-MB-231 n = 4, p = 0.0006, MDA-MB-468 n = 9, p = 2.33E-06, SkBr3 n = 4, p = 0.0004, NDL n = 4, p = 0.0344, MCF7 n = 10, p = 1.54E-08) d MCF7, MDA-MB-231, MDA-MB-468, Met-1, and NDL cells stimulated with Vector- or Wnt5a-conditioned media for 1 h blotted for Dvl2. e MDA-MB-231, SkBr3, T47D, and MCF7 cells stimulated with Vector- or Wnt5a-conditioned media for 1 h blotted for phospho-Vangl2 and Vangl2. f MDA-MB-231 cells stably expressing Control, shVangl2-1, or shVangl2-2 blotted for Dvl2. g Relative cell migration quantification of MDA-MB-231 cells stably expressing Control, shVangl2-1, or shVangl2-2 stimulated with Vector- or Wnt5a- conditioned media (control: vector- vs Wnt5a-conditioned media n = 4, p = 0.0003, Control vs shVangl2-1 + vector-conditioned media n = 4, p = 0.0003, control vs shVangl2-2 + vector-conditioned media n = 4, p = 0.0003, shVangl2-1: vector- vs Wnt5a-conditioned media n = 4, p = 0.7256, shVangl2-2: vector- vs Wnt5a-conditioned media n = 4, p = 0.5804). Bar graphs represent the mean ± sem of experimental replicates (n). Significance was determined by a two-sided unpaired t test with Welch’s correction, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Vangl1 and Vangl2 overexpression promote breast cancer cell migration and potentiate Wnt/PCP signaling. a, b Representative brightfield images of migrating NDL cells stably expressing Vector, Vangl1, or Vangl2 at 0 and 12 h, scale bar = 200 µm (a) and leading-edge dynamics at 12 h, scale bar = 50 µm (b). c Relative cell migration quantification of BT549, Met-1, NDL and MCF7 cells stably expressing Vector or Vangl1 (BT549 n = 5, p = 0.0001, Met-1 n = 3, p = 0.0339, NDL n = 6, p = 0.0108, MCF7 n = 7, p = 0.0035,). d Relative cell migration quantification of BT549, MDA-MB-231, Met-1, NDL, and MCF7 cells stably expressing Vector or Vangl2 (BT549 n = 3, p = 0.0210, MDA-MB-231 n = 3, p = 0.0050, Met-1 n = 3, p = 0.0412, NDL n = 8, p = 0.0049, MCF7 n = 8, p = 6.17E-05). e–f NDL and Met-1 cells stably expressing Vector, Vangl1 or Vangl2 blotted for Dvl2 (e) and quantification of Dvl2 phosphorylation in NDL and Met-1 cells stably expressing Vector, Vangl1 or Vangl2 (f) (NDL-Vangl1 n = 3, p = 0.0291, NDL-Vangl2 n = 6, p = 0.0453, Met-1-Vangl1 n = 5, p = 0.0009, Met-1-Vangl1 n = 3, p = 0.0119). g, h NDL cells stably expressing Vector, Vangl1, or Vangl2 treated with DMSO or 100 nM C59 for 24 h blotted for Dvl2 (g) and quantification of relative Dvl2 phosphorylation (h). Bar graphs represent the mean ± sem of experimental replicates (n). Significance was determined by a two-sided unpaired t test with Welch’s correction, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Wnt/PCP signaling drives collective cell invasion ex vivo and is upregulated in the K14-positive leader cell population. a Representative bright field images of MMTV-PyMT-derived mouse mammary tumor organoids stably overexpressing Vector, Vangl1, Vangl2 or Wnt5a invading into collagen in the presence of 2.5 nm bFGF, scale bars = 50 µm. b Quantification of the percentage of organoids counted with 0–2, 3–5, and 6 + collectively invading protrusions for Vector-, Vangl1-, Vangl2-, or Wnt5a-expressing organoids (Vector n = 547, Vangl1 n = 371, Vangl2 n = 276, Wnt5a n = 456 organoids counted from six independent experiments, p values represent Vector (V) vs Vangl1 (V1), Vangl2 (V2), Wnt5a (W), 0–2 protrusions; V vs V1 p = 0.0011, V vs V2 p = 6.32E-06, V vs W p = 2.50E-05, 3–5 protrusions; V vs V1 p = 0.0001, V vs V2 p = 8.94E-05, V vs W p = 0.0092, 6 + protrusions; V vs V1 p = 0.0027, V vs V2 p = 0.0132, V vs W p = 0.0024). Bar graphs represent the mean ± sem of experimental replicates (n), significance was determined by a two-sided unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Analysis of RNA-sequencing data set SRP066316 from NCBI Sequence Read Archive for Wnt5a, Vangl2, and Vangl1 transcript in K14- and K14 + cells derived from MMTV-PyMT tumors (Wnt5a p = 7.15E-05, Vangl2 p = 0.0049, VANGL1 p = 0.0422), significance was determined by likelihood ratio test followed by Benjamin–Hochberg correction for multiple hypothesis testing

Fig. 4

Vangl localizes to the migratory protrusions of single migratory cells and the leading edge of leader cells in collectively migrating breast cancer cells and promotes a hyper-protrusive morphology. a Representative confocal images of migratory MDA-MB-231 cells stained for Vangl1:green or Vangl2:green, Actin: gray, and DAPI: blue. b Representative confocal images of collectively migrating MCF7-Vector cells stained for Vangl1: green, Actin: gray, and DAPI: blue, MCF7-Flag-Vangl1 cells stained for Flag: green, Actin: gray, and DAPI: blue, and MCF7-V5-Vangl2 cells stained for V5: green, Actin: gray, and DAPI: blue, yellow arrows: Vangl-rich protrusions, scale bar = 10 µm

Fig. 5

Vangl2 regulates RhoA activity in leader cells of collectively migrating breast cancer cells. a Representative spatial activity profiles of RhoA in collectively migrating MCF7 cells stably expressing RhoA-FRET biosensor and Control (top row), shVangl2-1 (middle row), or shVangl2-2 (bottom row) at 1 h (left column), 6 h (center column), and 12 h (right column. Color bars indicate the range of RhoA-FRET biosensor ratios. Scale bar = 25 µm. b RhoA activity as a function of distance in µm from the leading edge of collectively migrating MCF7 cells stably expressing Vector (n = 27 wells), shVangl2-1 (n = 24 wells), or shVangl2-2 (n = 25 wells) at 1, 6, and 12 h of migration, error bars indicate ± sem. c, d RhoA activity after 1 h of migration at 5 µm (c) and 100 µm (d) from the leading edge of collectively migrating MCF7 cells stably expressing Vector, shVangl2-1 (Vector vs shVangl2-1, 5 µm p = 0.00388, 100 µm p = 0.1010, or shVangl2-2 (Vector vs shVangl2-2, 5 µm p = 0.0384, 100 µm p = 0.1309), significance was determined by a two-sided unpaired t test

Fig. 6

Vangl2 deletion suppresses mammary tumor metastasis to the lung. a Representative images of formalin-fixed, paraffin-embedded sections from Vangl2^+/+/NDL and Vangl2^fl/fl/NDL lung tissue following immunodetection of ErbB2 (top panel) and H&E staining (bottom panel). Examples of ErbB2-positive metastatic lung lesions are denoted by black arrowheads, scale bar = 500 µm. b–d Lung lobes (5 lobes per mouse) were evaluated by histology for the occurrence of metastatic lesions for Vangl2^+/+/NDL (n = 20) and Vangl2^fl/fl/NDL (n = 20) tumor-bearing animals. The number of mice bearing metastatic lesions (b), numbers of metastatic lesions (c), and metastatic burden (d) were assessed. e–g Vangl2/NDL primary tumor growth characteristics were assessed for Vangl2^+/+/NDL (n = 20) and Vangl2^fl/fl/NDL (n = 20) tumor-bearing animals, including number of palpable tumors (e), total tumor volume (f), and average tumor volume (g). Significance was determined by Mann–Whitney test and bar graphs represent the mean ± sem of experimental replicates (n)