S100P interacts with integrin α7 and increases cancer cell migration and invasion in lung cancer

Abstract

S100P, a Ca2+ binding protein, has been shown to be overexpressed in various cancers. However, its functional character in lung cancer remains largely unknown. In this study, we show that S100P increases cancer migration, invasion and metastasis in lung cancer cells. Ectopic expression of S100P increases migration, invasion and EMT in less invasive CL1-0 lung cancer cells. Conversely, knockdown of S100P suppressed migration and invasion, and caused a reversion of EMT in highly invasive lung cancer cells. These effects were transduced by increasing the interaction of S100P with integrin α7, which activated focal adhesion kinase (FAK) and AKT. Blocking FAK significantly decreased S100P-induced migration by decreasing Src and AKT activation, whereas inhibiting AKT reduced S100P upregulation on ZEB1 expression. Further study has indicated that S100P knockdown prevents the spread of highly metastatic human lung cancer in animal models. This study therefore suggests that S100P represents a critical activator of lung cancer metastasis. Detection and targeted treatment of S100P-expressing cancer is an attractive therapeutic strategy in treating lung cancer.

Keywords: FAK; S100P; ZEB1; metastasis; oncogene.

Figure 1. Elevated S100P expression in highly invasive lung cancer cells and tumor regions

A. The S100P level in nontumorous and tumorous regions of lung cancer patients. B. The mRNA transcript of S100P in highly invasive CL1-5 and less invasive CL1-0 cells. Sections of nontumorous and tumorous regions of lung cancer patients were co-stained by anti-S100P antibody and DAPI. S100P mRNA levels were determined by qRT-PCR. Data were shown as the mean±SD. *p < 0.05, or a significant difference between the two test groups.

Figure 2. Loss of S100P protein caused MET and decreased cell migration and invasion in lung cancer cells

A. Transfection of S100P shRNA plasmid decreased S100P. B. Morphologic imaging of S100P knockdown CL1-5 and A549 cells. C. Expression of the EMT markers as analyzed by immunoblot. S100P knockdown decreased cell migration D. and invasion E.. Protein levels were assessed by immunoblot. Migratory or invasive cells were stained by crystal violet or quantitated by a fluorescence dye. Data were shown as the mean±SD. *p < 0.05, or a significant difference between the two test groups.

Figure 3. Overexpression of S100P protein caused EMT and increased cell migration and invasion

A. The expression of S100P in CL1-0 and A549 cells. B. Morphologic change of S100P overexpressing CL1-0 and A549 cells. C. Expression of EMT markers. Ectopic expression of S100P increased cancer migration D. and invasion E.. The levels of various proteins were measured by immunoblot. Cell migration and invasion were determined as described above. Data were shown as the mean±SD. *p < 0.05, or a significant difference between the two test groups.

Figure 4. S100P-mediated EMT by ZEB1

The expression of EMT-related transcription factors in A. S100P-knockdown and B. overexpressing cells. C. Silencing of ZEB1 decreased cell migration in S100P overexpression cells. D. Inhibition of ZEB1 restored E-cadherin level in S100P overexpressing CL1-0. Ectopic expression of S100P CL1-0 cells was transfected with either control or ZEB1siRNA for 24 h. The cells were submitted to migration (trans-well assay) and immunoblot. Data were shown as the mean±SD. *p < 0.05, or a significant difference between the two test groups.

Figure 5. FAK/Src/AKT signaling is involved in S100P-mediated cancer

A. Inhibition of S100P decreased the activation of FAK, Src, and AKT. B. Ectopic expression of S100P increased the phosphorylation of FAK, Src, and AKT. C. Inhibition of FAK by siRNA transfection prevented S100P-mediated cell migration. D. Knockdown of AKT by siRNA decreased S100P-mediated cell migration. E. Knockdown of FAK by siRNA reduced AKT and Src activation in S100P overexpressing CL1-0 cells. F. Transfection of AKT siRNA decreased ZEB1 upregulation in S100P overexpressing CL1-0 cells. The cells were submitted to migration (transwell assay) and immunoblot. The expressions of various proteins were assessed by immunoblot. Data were shown as the mean±SD. *p < 0.05 or significant difference between control and test groups.

Figure 6. Integrin α7 was involved in S100P-mediated cancer progression

A. The interaction of S100P with integrin α7. B. The efficacy of integrin α7 siRNA transfection. C. Knockdown of integrin α7 decreased S100P-mediated cell migration. D. Inhibition of integrin α7 reduced N-cadherin upregulation and FAK in S100P overexpressing CL1-0 cells. E. The expression of integrin α7 in CL1-0 and CL1-5 cells. Levels of integrin α7 were determined using anti-integrin α7 antibody. The cells were submitted to migration (transwell assay) and immunoblot. The expressions of various proteins were assessed by immunoblot. Data were shown as mean±SD. *p < 0.05 or significant difference between control and test groups.

Figure 7. Inhibition of S100P decreased lung cancer metastasis in vivo

A. The lung tumor nodules of mice. B. H&E staining of lung tumor sections. C. The phosphorylation of FAK and AKT in lung tumors of mice. *p < 0.05 or significant difference in control shRNA plasmid vs. S100P shRNA plasmid, as analyzed by Student's t test.