Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion

Abstract

Background: LKB1, also known as STK11, is a master kinase that serves as an energy metabolic sensor and is involved in cell polarity regulation. Recent studies have indicated that LKB1 is related to breast tumorigenesis and breast cancer progression. However, little work has been done on the roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer. In this study, we tried to prove that loss of LKB1 disrupts breast epithelial cell polarity and causes tumor metastasis and invasion.

Methods: The relationships of LKB1 expression to clinic-pathological parameters and epithelial markers E-cadherin and high-molecular-weight -cytokeratin (HMW-CK) were investigated in 80 clinical breast cancer tissue samples and their paired normal control breast tissue samples by using immunohistochemistry. Then, the LKB1 expressions in metastatic and non-metastatic breast cancer cell lines were compared. The roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer were determined by using immunofluorescence, western blot assay, and cell migration and invasive assays. Finally, the non-transformed human breast cell line MCF-10A was cultured in three dimensions to further reveal the role of LKB1 in breast epithelial cell polarity maintenance.

Results: Histopathological analysis showed that LKB1 expression level was significantly negatively correlated with breast cancer TNM stage, and positively correlated with ER/PR status and expression levels of E-cadherin and HMW-CK. Immunofluorescence staining showed that LKB1 was co-localized with E-cadherin at adheren junctions. In vitro analysis revealed that loss of LKB1 expression enhanced migration, invasion and the acquisition of mesenchymal phenotype, while LKB1 overexpression in MDA-MB-435 s cells, which have a low basal level of LKB1 expression, promoted the acquisition of epithelial phenotype. Finally, it was found for the first time that endogenous LKB1 knockdown resulted in abnormal cell polarity in acini formed by non-transformed breast epithelial cells grown in 3D culture.

Conclusion: Our data indicated that low expression of LKB1 was significantly associated with established markers of unfavorable breast cancer prognosis, such as loss of ER/PR, E-cadherin and HMW-CK. Knockdown of endogenous LKB1 gave rise to dysregulation of cell polarity and invasive phenotype of breast cancer cells.

Figure 1

LKB1 immunohistochemistry staining in normal mammary and breast cancer tissues. Low-power (scale bar, 150 ?m) and high-power (scale bar, 50 ?m) photomicrographs showed (A) LKB1 localization in the cytoplasm of normal luminal epithelial cells, (B) strong LKB1 immunostaining in stageIbreast cancer tissues, (C) moderately positive LKB1 staining in stage IIbreast cancer tissues, (D) weakly positive LKB1 staining in stage III breast cancer tissues, (E) strong LKB1 immunostaining in ER/PR+++ breast cancer tissues, (F) weakly positive LKB1 immunostaining in ER/PR?+?breast cancer tissues, (G) strong LKB1 immunostaining in E-cadherin+++ breast cancer tissues, (H) negative LKB1 staining in E-cadherin breast cancer tissues, (I) strong LKB1 immunostaining in HMW-CK+++ breast cancer tissues, and (J) negative LKB1staining in HMW-CK breast cancer tissues.

Figure 2

The relationship of LKB1 expression level with cancer stage, ER/PR status and HMW-CK expression. Distribution of breast cancer cases showing the relationship of LKB1 expression level with cancer stage (stage I/stage II/stage III) (A), with (B), with E-cadherin expression (C), and with HMW-CK expression (D).

Figure 3

LKB1 expressions in different breast cancer cell lines. (A) LKB1 expressions in immortalized breast epithelial cell line MCF-10A, luminal A subtype (MCF-7, BT474 and T47D) and metastatic SKBR3 and MDA-MB-435 s. (B) LKB1 expressions were quantified using scanning densitometry and LKB1 expressions in breast cancer cells were calculated as ratios to the LKB1 expression in immortalized breast epithelial cell line MCF-10A.

Figure 4

LKB1 affected migration and invasion of breast cancer cell lines. (A) LKB1 protein levels in control non-targeting siRNA (NT) and siLKB1 1342/1972 transfected MCF-7 cells were detected by Western blot. LKB1 knockdown efficiency was quantified using densitometry. (B) Loss of LKB1 increased cell migration in wound healing assay. Cell migration into the wound area was photographed at indicated time points, and the distance between the edges of the lesion was calculated as the migration distance (mm). (C) Loss of LKB1 increased cell invasion in matrigel transwell chambers. Migrating cells were stained and photographed. Invasion properties were expressed as the number of cells per mm².

Figure 5

LKB1 was localized at adheren junctions and regulated the expressions of EMT markers. (A) MCF-10A was stained for LKB1 (green), E-cadherin (red) and DAPI (blue). (B) Expressions of EMT markers in control and LKB1 overexpressing MDA-MB-435 s. (C) MCF-7 cells were transfected with non-targeting siRNA (NT) or siLKB1(1342/1972). The protein levels of E-cadherin, N-cadherin and ?-SMA were determined by western blot.

Figure 6

E-cadherin mRNA expression was down-regulated in LKB1 knock-down cells. MCF-7 cells were transfected with non-targeting siRNA (NT) or siLKB1(1342/1972). The mRNA levels of E-cadherin were detected by RT-RCR.

Figure 7

Loss of LKB1 resulted in morphological alterations and dysregulation of cell polarity in 3D cultures. (A) MCF-10A cells were transfected with non-targeting siRNA (NT) or siLKB1(1342/1972). The protein levels of LKB1 were detected by western blot. Efficiency of knockdown was quantified using densitometry. (B) Morphogenesis of MCF-10A cells plated on matrigel. Phase images of MCF-10A cells were taken on Day 4 (scale bar, 100 ?m). A single acinar structure was taken on Day 8 (scale bar, 100 ?m). (C) non-targeting siRNA (NT) or siLKB1(1342/1972) transfected MCF-10A cells stained with E-cadherin (red) on Day 16. (D) non-targeting siRNA (NT) or siLKB1(1342/1972) transfected MCF-10A cells stained with GM130 (red, Golgi marker) on Day 12.