Distinct expression profiles and functions of Kindlins in breast cancer

Abstract

Background: Kindlin-1, - 2, and - 3 are the three members of the Kindlin family. They are best known as regulators of integrin functions, contributing to fundamental biological processes such as cell survival, adhesion and migration. Their deregulation leads to diverse pathologies including a broad range of cancers in which both, tumor-promoting and tumor-inhibiting functions have been described.

Methods: To better characterize Kindlins implication in breast cancer, in vitro experiments were performed in a series of cancer cell lines. We first assessed their expression profiles and subcellular distributions. Then, their involvement in breast cancer cell morphology, migration and invasion was verified by examining phenotypic changes induced by the depletion of either isoforms using RNA interference. An expression study was performed in a series of breast cancer patient derived xenografts (n = 58) to define the epithelial and stromal contribution of each Kindlin. Finally, we analyzed the expression levels of the three Kindlins in a large series of human breast tumors, at the RNA (n = 438) and protein (n = 129) levels and we evaluated their correlation with the clinical outcome.

Results: We determined that Kindlin-1 and Kindlin-2, but not Kindlin-3, were expressed in breast tumor cells. We uncovered the compensatory roles of Kindlin-1 and -2 in focal adhesion dynamics and cell motility. Remarkably, Kindlin-2 had a predominant effect on cell spreading and Kindlin-1 on cell invasion. In line with these experimental observations, Kindlin-1 overexpression was associated with a worse patients' outcome. Notably, Kindlin-3, expressed by tumor infiltrating leukocytes, also correlated with a poor prognosis of breast cancer patients.

Conclusion: This study demonstrates that each one of the Kindlin family members has a different expression profile emphasizing their redundant and complementary roles in breast tumor cells. We highlight the specific link between Kindlin-1 and breast cancer progression. In addition, Kindlin-3 overexpression in the tumor microenvironment is associated with more aggressive breast tumors. These results suggest that Kindlins play distinctive roles in breast cancer. Kindlins may be useful in identifying breast cancer patients with a worst prognosis and may offer new avenues for therapeutic intervention against cancer progression.

Keywords: Breast cancer; Invasion; Kindlins; Prognosis; Redundant functions.

Fig. 1

Kindlin-1, −2 and − 3 expression, involvement in morphology and subcellular localization in breast cancer cells. a Western Blots were performed in order to compare protein levels of Kindlin-1, − 2 and − 3 between different breast cancer cell lines and a hematopoietic cell line (THP1). (b-d) MD-MB-231, BT20 and MDA MB 468 cells were transfected with control siRNA (si-ctrl), KIND1 siRNA (si-Kind1), KIND2 siRNA (si-Kind2) alone or in combination (si-Kind1 + si-Kind2) for five days. b Cellular extracts were immunoblotted with anti-Kindlin-1, anti-Kindlin-2, and anti-GAPDH (loading control) antibodies. c Phase contrast microscopy was performed in MDA-MB-231 cells to calculate the roundness (% of rounded cells) and cell area (μm²) represented as the mean +/− SEM of values. Statistical analysis were made by performing a t-test (****p < 0.0001; ***p < 0.001; *p < 0.05; ns: not significant). d Five days after transfection, MDA-MB-231 cells were also fixed, permeabilized and immunostained with anti-Kindlin-1 and anti-Kindlin-2 antibodies. Cells were then counterstained with DAPI and imaged with a fluorescence microscope (original magnification: X100)

Fig. 2

Kindlin-1 and Kindlin-2 involvement in breast cancer cell motility. MD-MB-231 cells were transfected with control siRNA (si-ctrl), KIND1 siRNA (si-Kind1), KIND2 siRNA (si-Kind2) alone or in combination (si-Kind1 + si-Kind2) for five days. a Time-lapse imaging was performed for 24 h. Images show representative trajectories travelled by cells during 180 min. b Plots show overlays of the representative trajectories travelled by cells .Velocity was quantified and represented as the mean +/− SEM of values (n = 20 cells tracked by condition). Statistical analysis were performed by a t-test (****p < 0.0001; **p < 0.01; *p < 0.05). Results are representative of experiments performed at least in duplicate. c A transwell cell invasion assay was performed for 24 h. Cells were then counterstained with DAPI and imaged with a fluorescence microscope. The number of invasive cells was quantified and represented as the mean ± SEM of values. Statistical analyses were performed by a t-test (*p < 0.05; ns: not significant). A representative image of three independent experiments is shown for each condition. Scale bar = 50 μm

Fig. 3

Kindlins expression is cell-type specific. a Immunohistochemical staining of the normal mammary gland were performed in different patient’s samples to analyze the levels and localizations of these proteins (En: endothelial cells, F: fibroblasts, H: hematopoietic cells, Lu: luminal epithelial cells, Ba: basal epithelial cells). b Establishment of breast cancer patient-derived xenografts (PDX): Primary breast tumor fragments derived from patients are engrafted into immunocompromised mice. Tumors can be implanted into the interscapular fat pad, the mammary fat pad or in the flank. Xenografts appear at the graft site 1–12 months after grafting, they are subsequently transplanted from mouse to mouse; adapted from [44]. Then, Kindlins transcript levels were assessed in bulk tumors by using species-specific primers (mean ± SE values are represented)

Fig. 4

Kindlin-1, − 2 and − 3 protein expressions in breast cancer and adjacent noncancerous tissue. a Representative images of Kindlin-1, − 2 and − 3 immunohistochemical staining in breast cancer TMA and normal adjacent tissues. b Scatter plot showing range of Kindlin-1 and -2 expressions related to histology. Each point represents the H-score from a single tissue sample ranging from total absence of Kindlin in the epithelial compartment (H-score 0), to very strong Kindlin staining (H-score 300). Mean H-score ± SE represented. Statistical differences between normal and tumor expression levels were performed using the Mann-Whitney test

Fig. 5

Kindlin-1 expression correlates with lung metastasis. a Box-and-Whisker plot showing kindlin-1, − 2 and − 3 mRNA expression levels in a series of 438 breast cancer patients grouped according to four well described breast tumor molecular subtypes (Triple negative, ERBB2, Luminal A and Luminal B). Statistical analysis were made by performing Tukey’s multiple comparison test (***p < 0.001; **p < 0.01; *p < 0.05). b Kaplan-Meier curves showing lung and bone metastasis-free survival of patients with tumors expressing high (red lines) vs. low (blue lines) levels of Kindlin-1, − 2 and − 3 analyzed by qRT-PCR. Statistical analyses were performed by a Log-rank test