Receptor-type Protein tyrosine phosphatase β regulates met phosphorylation and function in head and neck squamous cell carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met) pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β). We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP) kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

Figure 1

RPTP-β is downregulated in metastatic HNSCC cell lines. Total RNA and whole-cell lysates were prepared from HOKCs, cells from primary HNSCC tumors (UMSCC-17A and UMSCC-22A), and cells from metastatic HNSCC tumors of the same patients (UMSCC-17B and UMSCC-22B). RPTP-β mRNA and internal control housekeeping gene 36B4 were quantified by real-time reverse transcription-PCR. RPTP-β mRNA was not detectable in cells from secondary metastatic tumors. Inset shows representative Western blot of RPTP-β protein, with β-actin used as loading control. N = 3.6, *P < .05 versus HOKCs.

Figure 2

RPTP-β regulates Met tyrosine 1356 phosphorylation in HNSCC cells. (A) UMSCC-22B metastatic HNSCC tumor cells were infected with empty adenovirus or RPTP-β-expressing adenovirus. (B) UMSCC-22A primary HNSCC tumor cells were infected with NT or RPTP-β-targeting shRNA lentivirus. Forty-eight hours post-infection, cells were treated with vehicle (-) or HGF (10 ng/ml, +) for 30 minutes. Equal amounts of whole-cell lysates were analyzed by Western blot probed with indicated antibodies. Immunoreactive bands were quantified by chemifluorescence using a STORM Image analyzer. Data are means ± SEM, N = 3; *P < .05. Inset shows representative Western blots.

Figure 3

RPTP-β regulates HGF-induced Grb2 binding to Met and downstream activation of MEK and ERK in HNSCC cells. (A) UMSCC-22B metastatic HNSCC tumor cells were infected with empty adenovirus or RPTP-β-expressing adenovirus. Forty-eight hours post-infection, cells were treated with vehicle (-, Ctrl) or HGF (10 ng/ml, +) for 15 minutes. Met was immunoprecipitated from whole-cell lysates, and immunoprecipitates were analyzed for Grb2 and Met by Western blot. (B and C) UMSCC-22B cells were treated as in A, except HGF treatment time was 30 minutes, and analyzed for MEK phosphorylation (B) and ERK phosphorylation (C). (D and E) UMSCC-22A primary HNSCC tumor cells were infected with NT or RPTP-β-targeting shRNA lentivirus. UMSCC-22A cells were treated as in A, except HGF treatment time was 30 minutes, and analyzed for MEK phosphorylation (D) and ERK phosphorylation (E). Immunoreactive bands were quantified by chemifluorescence using a STORM Image Analyzer. Data are means ± SEM, N = 3; *P < .05. Insets show representative Western blots.

Figure 4

RPTP-β regulates HGF-induced HNSCC cell migration. (A) UMSCC-22B metastatic HNSCC tumor cells were infected with empty adenovirus or RPTP-β-expressing adenovirus. (B) UMSCC-22A primary HNSCC tumor cells were infected with NT or RPTP-β. targeting shRNA lentivirus. Forty-eight hours post-infection, confluent cells were treated with 10 µg/ml mitomycin for 30 minutes and scratched with a pipette tip to create a cell-free zone. Cells were then incubated with or without HGF (10 ng/ml) for 48 hours. A representative image of three independent experiments is shown.

Figure 5

RPTP-β inhibits proliferation of metastatic tumor-derived HNSCC cells. UMSCC-22B cells were infected with empty adenovirus or RPTP-β adenovirus. Cells were harvested at the indicated times post-infection and counted with a hemocytometer. Results are means ± SEM. N = 6; *P < .05.

Figure 6

RPTP-β regulates HGF-induced HNSCC cell invasion. (A) UMSCC-22B cells were infected with empty or RPTP-β-expressing adenovirus. (B) UMSCC-22A cells were infected with NT or RPTP-β-targeting shRNA lentivirus. Forty-eight hours post-infection, cells (1.5 x 10⁵ cells/well) were harvested and seeded inmatrigel invasion assay wells in Dulbecco's modified Eagle's medium (with 2% FBS), with or without HGF (10 ng/ml) for 48 hours. Cells that passed through the matrigel were stained and photographed. Representative images from three independent experiments are shown. Cells stained purple. Matrigel stained yellow/orange.

Figure 7

RPTP-β and Met protein expression levels are altered in HNSCC tumors. Formalin-fixed paraffin-embedded HNSCC tumor sections and normal oral tissue sections were stained with control IgG, (A) RPTP-β or (B) Met antibodies by immunohistochemistry. Representative images of normal tissue from four individuals and HNSCC tumors from eight individuals are shown.