CXCL12/CXCR4 Promotes Laryngeal and Hypopharyngeal Squamous Cell Carcinoma Metastasis by MMP-13-Dependent Invasion via

ERK1,2/AP-1 Pathway

Ching-Ting Tan1, Chia-Yu Chu 2,3, Ying-Chang Lu 1,3, Hsaio-Hui Wu1, Jenq-Yuh Ko1 and Min-Liang Kuo3

1 Department of Otolaryngology, 2 Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine,

Taipei, Taiwan3 Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine and Angiogenesis Research Center, National Taiwan

University, Taipei, Taiwan

IntroductionIntroductionCancer of the larynx and hypopharynx is one of the most common head and neck malignancy. Squamous cell carcinoma (SCC) is the most common histopathologic type of the laryngeal and hypopharyngeal malignancies, and lymph node metastasis is frequently seen in laryngeal and hypopharyngeal SCC and it directly contributes to the prognosis of patients with laryngeal and hypopharyngeal SCC. In spite of manyadvances in surgery and cancer therapy in recent years, there was no significant improvement in survival rates of head and neck squamous cell carcinoma patients. Therefore, understanding the detailed mechanism by which LHSCC metastasize to local lymph node is of importance and urgently needed. In this report, we demonstrated that LHSCC express high levels of functional CXCR4 receptor, which is a native receptor for the chemokine stromal-cell-derived factor-1 (SDF-1/CXCL12). Immunohistochemical analysis of primary tumor samples from LHSCCpatients revealed significant expression of both CXCR4 and CXCL12. The higher expression of CXCR4 is correlated with the patients who have lymph node metastasis and distant metastasis. Reverse transcriptase-polymerase chain reaction and Western blot examination demonstrated that the CXCR4 mRNA and protein were expressed in LHSCC cell lines as well. However, the expression of CXCL12 mRNAwas not observed in LHSCC cell lines. We further showed that CXCL12 treatment enhanced the activation of ERK pathway and the motility/invasion abilities of LHSCC cell lines and that were blocked by treatment with CXCR4 antagonist (AMD3100) and specific MEK inhibitor (U0126). Since MMPs are critically involved in cell invasiveness, we thus examined which MMP is possibly involved in the CXCL12-induced chemoinvasion of LHSCC cells. In this study, we investigated the possible role of CXCL12/CXCR4 axis and its related signaling in LHSCC. Our results show that LHSCC not only highly express CXCR4 but also its ligand, CXCL12. Treatment with CXCL12enhanced LHSCC cell migration and chemoinvasion. RT-PCR and Western blot analysis showed that MMP-13 was a downstream effector gene of CXCL12/CXCR4 signaling. In addition, the up-regulation of MMP-13 was mediated by Erk1/2 pathway and subsequent transcriptional factor AP-1.

MethodsMethods1. RNA extraction and RT-PCR assay2. Migration and Invasion assay 3. Western blotting and Immunohistochemistry staining 4. Nuclear and cytosol protein separation

ResultsResults

Fig.1 Relationship between CXCL12/CXCR4 Expression and Clinicopathological Findings in LHSCC.

CXCR4 CXCL12

I III

IIIV

Laryngealcancer

Hypopharyngealcancer

Fig.2 CXCL12 Enhanced Migration and Invasion of CXCR4-Positive LHSCC Cells

CXCR4

CXCL12

GAPDH

P’t

FaDu

CM SF

HEp-2

CM SF

HEp-2 FaDu

CXCR4

β-actin

CM SF CM SF

Fig.3 ERK1/2 Pathway is Essential for the CXCL12/CXCR4 Function in LHSCC

0 5 15 30 60 (min)

p-ERK

p-AKT

ERK1

FaDu

0 5 15 30 60 (min)

p-ERK

ERK1

p-AKT

Fig.4 Upregulation of MMP-13 by CXCL12 Involved in Invasion of LHSCC Cells

CXCL12

0 2 4 8 16 24 (hr)

MMP-2

MMP-9

MMP-13

GADPH

CXCL12

0 2 4 8 16 24 (hr)

MMP-13

β-actin

ConclusionConclusionCXCL12 enhances LHSCC cell invasion through paracrinally activates CXCR4 and in turn triggering an ERK/c-Jun-dependent MMP-13 up-regulation. It provide new information regarding signaling pathways that may regulate CXCL12-induced metastasis in LHSCC.

MMP-13

GAPDH

MMP13

β-actinCXCL12 c-Jun antisensec-Jun sense

---

-+-

++ -

--+

+ -+

+ --

p-c-JunSP-1

α-tubulin

NucleusSDF-1 α 0 10 30 60 120 240 (min)

Cytosol

0 10 30 60 120 240 (min)

p-c-JunSP-1

α-tubulin

SDF-1 α

SDF-1 αc-jun antisense(μg)

c-jun sense(μg)

- + + + + +- - 1 3 5 -- - - - - 5

Fig. 5 ERK1,2/AP-1 Kinase Pathway Is Involved in CXCL12 Modulation of MMP-13-Mediated LHSCC Cell Invasion

GAPDH

CXCL122 4 (hr)

MMP-13

U0126 0 0 20 0 10 20 (20μM)

MMP13

β-actin

p-ERK

CXCL12 - + + + + + (100 ng/ml)Lovastatin - - 0.1 1.0 10 - (μΜ)U0126 - - - - - 20 (μΜ)

MMP-13

GAPDH

β-actin

MMP-13

CXCL12 - + + + + (100 ng/ml)LY294002 - - - - 20 (μM)U0126 - - 10 20 - (μM)

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