LAMB3 mediates metastatic tumor behavior in papillary thyroid cancer by regulating c-MET/Akt signals

Laminin subunit beta-3 (LAMB3) encodes one of the three subunits of LM-332, a protein of the extracellular matrix secreted by cultured human keratinocytes. While LAMB3 is involved in the invasive and metastatic abilities of several tumor types, including those found in the colon, pancreas, lung, cervix, stomach, and prostate, its mechanism of action in thyroid cancer has not been investigated previously. Our results show that LAMB3 is up-regulated in papillary thyroid cancer, and that its suppression reduces cell migration/invasion via down-regulation of epithelial‒mesenchymal transition-associated proteins (N-cadherin, vimentin, slug) and inhibition of matrix metalloproteinase 9. LAMB3 suppression also significantly decreases Akt phosphorylation and inhibits the transcription of c-MET, reducing its activation. These results suggest that LAMB3 leads to tumor invasion via Akt activation induced by the HGF/c-MET axis in papillary thyroid cancer cells. Our findings reveal a novel mechanism of action for LAMB3 in papillary thyroid cancer cells.

Cell migration and invasion assay. Transwell membranes (24-well, Costar, Cambridge, MA, USA) were either coated with Matrigel for 6 h for invasion assays or were used without Matrigel for migration assays. Cells (0.5 × 10 5 ) in serum-free medium were seeded into each upper chamber, and 600 μl medium supplemented with 10% FBS were added to each lower chamber. After incubation for 24 h, cells adhering to the upper surface of the membrane were removed with a cotton swab. Cells that had invaded or migrated, which were adhered to the lower surface, were stained with 0.1% crystal violet and counted in four representative fields by light microscopy (200 × magnification). Zymography. TPC-1 cells (1 × 10 5 /well) cultured in 6-well plates were transfected with LAMB3 siRNA or negative control siRNA for 48 h. MMP activity was then measured in the conditioned culture media by substrate gel electrophoresis using 8% SDS-PAGE gels containing 0.2% gelatin. Conditioned medium samples were adjusted to equal protein concentrations, mixed with sample buffer (250 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 0.01% bromophenol blue), and loaded onto gels for protein separation by electrophoresis. To remove the SDS, the gels were soaked three times for 30 min at room temperature in Triton buffer (2.5% Triton X-100 in PBS). Gels were then incubated in Zymogram development buffer (#161-0766, Bio-Rad, Hercules, CA, USA) for 24 h at 37 °C and stained with EZblue reagent (Sigma-Aldrich, Louis, MO, USA, #G1041). Gels were destained to obtain clear bands, and quantitative results were obtained by densitometry.

Screening for RTK expression in cells.
RTKs were examined using a western blot array (Proteome Profiler Human Phospho-RTK Array Kit, ARY001B, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. All experiments were performed in duplicate. In brief, 1 × 10 7 cells/ml were washed in PBS and extracted using the appropriate kit buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich). Extracts were centrifuged, and the supernatants were diluted with sample buffer and applied to nitrocellulose membranes previously spotted with 49 anti-kinase receptor antibodies. RTKs were then detected using an HRP-conjugated anti-phospho-tyrosine antibody and chemiluminescence.
Statistical analysis. All in vitro experiments were repeated three times, and statistical significance was analyzed using two-sided Student's t-test. Data are presented as means ± standard deviation (SD), and a P value < 0.05 was considered statistically significant (*P < 0.05).

LAMB3 expression in thyroid cancer patient tissues and cell lines.
We first evaluated LAMB3 expression in normal and tumor tissues derived from the same thyroid cancer patients. Considerably higher expression of LAMB3 protein levels was detected in all five tumor samples relative to the normal tissues (Fig. 1A). The expression levels of LAMB3 mRNA and protein were also examined in a normal thyroid cell line (N-thy-ori) and in three PTC cell lines (B-CPAP, K1, and TPC-1). All PTC cell lines demonstrated notably higher LAMB3 expression at both the mRNA and proteins levels (Fig. 1B,C). The results indicate that overexpression of LAMB3 is correlated with tumorigenesis in PTC. We also examined the expression of LAMA3 and LAMC2 in PTC cell lines because many studies revealed that these could play an important role in carcinogenesis of several cancers (Fig. 1B). LAMC2 protein levels were increased in PTC cell lines compared to normal thyroid cell line (N-thyori). However, in our study we just focused on the regulation and functions of LAMB3 in PTCs.

LAMB3 promotes cell migration and invasion, but not proliferation, in PTC cells.
We hypothesized that LAMB3 participates in PTC tumorigenesis and metastasis. To investigate the functional significance and mechanism of action of LAMB3 in PTC, we used two PTC cell lines, TPC-1 (carrying RET/PTC-1) and B-CPAP (carrying BRaf V600E ). We first examined the effect of LAMB3 on cell proliferation. TPC-1 and B-CPAP cells were transiently transfected with LAMB3 siRNA or a negative control siRNA for 48 h, and cell proliferation was detected by WST-1 assay. LAMB3 knockdown specifically down-regulated LAMB3 but had no significant effect on cell proliferation in the two PTC lines ( Fig. 2A,B). These results suggest that LAMB3 is not associated with cell proliferation in PTC cells. Cell migration and invasion have been recognized as key steps in tumor metastasis. To determine the effects of LAMB3 on cell migration and invasion, TPC-1 and B-CPAP cells were transiently transfected with LAMB3 siRNAs or with negative control siRNA for 48 h. PTC cells were then allowed to migrate for 24 h in Transwell chambers (cell migration) or for 48 h in chambers with Matrigel (cell invasion). Although it had no effect on cell proliferation, LAMB3 knockdown considerably suppressed the migration and invasion of TPC-1 and B-CPAP cells (Fig. 2C-F). These results clearly show that LAMB3 promotes the migration and invasion of PTC cells.

LAMB3 regulates epithelial-mesenchymal transition (EMT)-related proteins and metastasisrelated proteins. The association between EMT and cell invasion has been demonstrated in cancer progres-
sion. To investigate whether LAMB3 promotes EMT, we examined both epithelial and mesenchymal markers, E-cadherin, N-cadherin, and vimentin, as well as the well-known EMT marker Slug, by immunoblot analysis. LAMB3 knockdown caused a significant decrease in N-cadherin, vimentin and Slug levels and increase in E-cadherin in TPC-1 and B-CPAP cells (Fig. 3A-D). Matrix MMPs, particularly MMP2 and MMP9, are integral to the processes of invasion and metastasis. To determine whether LAMB3 regulates MMPs, we investigated the expression and secretion of MMP2 and MMP9 in both PTC cell lines. Pro-MMP9 mRNA expression was significantly reduced by LAMB3 knockdown in TPC-1 and B-CPAP cells (Fig. 3E,F) and Pro-MMP9 secretion, analyzed by gelatin zymography was also inhibited in both cell lines (Fig. 3G). However, LAMB3 did not affect the expression or secretion of pro-MMP2. Taken together, these findings could suggest that LAMB3 induces the migration and invasion of PTC cells via EMT and MMP9 expression.
LAMB3 regulates PI3K-mediated Akt phosphorylation. We hypothesized that LAMB3 regulates cell migration and invasion via certain cellular signaling pathways. We investigated various pathways involved in tumorigenesis and/or metastasis. We observed that LAMB3 suppression significantly decreased Akt phosphorylation at serine 473, whereas the total level of Akt protein was not affected in TPC-1 and B-CPAP cells (Fig. 4A,B). PI3K/Akt signaling appears to play an important role in the progression of papillary cancers 21 . To elucidate whether Akt suppression reduces tumor cell migration in PTCs, a Transwell migration assay was performed using TPC-1 and B-CPAP cells following Akt inhibition by LY294002. Inhibition of PI3K/Akt signaling significantly suppressed TPC-1 cell migration (Fig. 4C,D). Akt inhibition by LY294002 also decreased levels of the EMT-related proteins vimentin, Slug, and Snail and increased that of the epithelial marker E-cadherin (Fig. 4E,F). The mRNA expression of MMP9 but not MMP2 was reduced by Akt inhibition (Fig. 4G,H). Collectively, these  results reveal that LAMB3 promotes the migration and invasion of PTC cells via activation of the PI3K/Akt signaling pathway, which leads to EMT and MMP9 activation.

LAMB3 leads to tumor invasion via Akt activation induced by the HGF/c-MET axis.
To identify RTKs activated by LAMB3 in TPC-1 cells, the activation status of 49 human RTKs was analyzed using a phospho-RTK array. Among these RTKs, phospho-c-MET was markedly decreased by LAMB3 knockdown (Fig. 5A). We validated the effect of LAMB3 knockdown on c-MET activation by western blot analysis. Total cell lysates were immunoblotted with antibodies for c-MET p-Y1003, Y1234/5, Y1359, and total c-MET. LAMB3 knockdown decreased c-MET phosphorylation as well as total c-MET levels in TPC-1 cells (Fig. 5B). Interestingly, the mRNA expression of c-MET was also reduced by LAMB3 knockdown, indicating that LAMB3 regulates the transcription of c-MET in TPC-1 and B-CPAP cells (Fig. 5C,D). We investigated whether Akt was regulated by c-MET under our conditions. Phospho-Akt levels were down-regulated in cells in which c-MET had been knocked down by siRNA, suggesting that c-MET acts upstream of Akt in TPC-1 and B-CPAP cells (Fig. 6A,B). We also investigated whether over-expression of c-MET reverses the effect of LAMB3 suppression on Akt activation, EMT/MMP-9 and migration/invasion. c-Met overexpression using co-transfection of vector attenuated the effect of LAMB3 suppression on not only migration/invasion (Fig. 6C-F), but also EMT related proteins (Fig. 6G,H) in both TPC-1 and B-CPAP cells. These results demonstrate that LAMB3 activates Akt via up-regulation of c-MET, and that this LAMB3/c-MET/Akt signaling cascade induces cell invasiveness in PTCs.

Discussion
Many studies have indicated that cell adhesion and extracellular matrix proteins contribute to the progression of cancer 22 . LM-332 (encoded by LAMA3, LAMB3, and LAMC2) has been linked to tumor invasiveness in various types of cancer 16,[23][24][25] . In particular, LAMB3, involved in the focal adhesion pathway, is a potential biomarker of cancer invasion and metastasis [16][17][18] . In our data evaluating the LAMB3 expression pattern in the human thyroid cancer tissues, all the tumor tissues had higher LAMB3 expression compared to normal tissues. Given that our study had relative small number of the cases and no localization data, wider-scale studies are mandatory including immunohistochemistry to confirm the expression pattern of LAMB3 and its clinical meaning. However, our study have strength in that to our best of knowledge, we evaluated for the first time the expression of LAMB3 in the thyroid carcinoma. Next, we found that although LAMB3 expression was relatively up-regulated in three PTC cell lines (TPC-1, B-CPAP, and K1), LAMB3 knockdown by siRNA had no significant effect on the proliferation of TPC-1 or B-CPAP cells. However, the migration and invasion of PTC cells were considerably reduced by LAMB3 knockdown. These data could suggest that LAMB3 expression in PTC cells is associated with stimulation of cell migration and invasion but is not significantly involved in cell proliferation.
The PI3K/Akt pathway has a fundamental role in thyroid tumorigenesis and represents an attractive target for pharmaceutical development for a variety of malignancies 9,10,26 . LAMB3 knockdown by siRNA considerably reduced the level of phospho-Akt without altering the level of total Akt protein. EMT is a critical process in tumorigenesis, tumor invasion, and metastasis, and refers to the molecular reprogramming and phenotypic changes that characterize the transition from polarized immotile epithelial cells to motile mesenchymal cells 27 . MMPs are important mediators of cellular events, including ECM degradation and remodeling, cell proliferation, apoptosis, cell invasion/migration, and morphological changes 28 . PI3K/Akt inhibition has been shown to block sonic hedgehog pathway-induced EMT, matrix MMP9 activity, and lymphangiogenesis, reducing tumor invasiveness and metastasis 29 . The expression of a constitutive nuclear form of FOXO1, which is inhibited by Akt signaling, significantly inhibited the MMP9 activation induced by EGF 30 . In this study, we confirmed that LAMB3 regulates EMT-related proteins and MMP9 via activation of Akt signaling.
To investigate the upstream component mediating Akt regulation by LAMB3, we focused on RTKs. Aberrant expression of certain RTKs has been implicated in the development and progression of many types of cancer. These RTKs have emerged as promising drug targets for cancer therapy 31 . c-MET is an RTK that, after binding to its ligand, hepatocyte growth factor, activates a wide range of different cellular signaling pathways, including those involved in cell proliferation, motility, migration, and invasion. Activation of the HGF/c-Met axis prevents apoptosis through activation of PI3K and subsequent Akt activation [12][13][14] . In a previous study, we found HGF/c-Met pathway activation to be associated with subclinical central neck lymph node metastasis of PTC 15 . Using a phospho-RTK array, we found that c-MET acts upstream of Akt, indicating that LAMB3 leads to tumor invasion via Akt activation induced by the HGF/c-MET axis in PTCs.
PTCs usually possess mutations in BRAF or RET/PTC genetic rearrangements. Many of the genetic changes observed, including RET/PTC gene rearrangements and RAS and BRAF mutations, lead to activation of the MAPK and PI3K/Akt signaling pathways 8 . As shown in our results, both TPC-1 cells (carrying RET/PTC-1) and B-CPAP cells (carrying BRaf V600E ) promoted tumor progression via LAMB3/Akt signaling, despite carrying different types of genetic alterations.
Recent reports have suggested that LAMB3 is a potential biomarker and drug target in PTC, and that monitoring several transcripts, including LAMB3, can afford high diagnostic accuracy 32,33 . In addition, our findings established that LAMB3 could promote the metastasis of PTC via activation of the PI3K/Akt pathway induced by the HGF/c-MET axis, leading to EMT and MMP9 activation. To our knowledge, this is the first report identifying a molecular mechanism of action for LAMB3 in PTC.
In conclusion, our findings could suggest that LAMB3 contributes to cancer cell migration and invasion in PTC via c-MET/Akt signaling. Elucidation of LAMB3-regulated cancer pathways may provide novel therapeutic strategies to control PTC metastasis.