TGFβ3-mediated induction of Periostin facilitates head and neck cancer growth and is associated with metastasis

The matrix-specific protein periostin (POSTN) is up-regulated in human cancers and associated with cancer growth, metastasis and angiogenesis. Although the stroma of cancer tissues is the main source of POSTN, it is still unclear how POSTN plays a role to facilitate the interplay between cancer cells and cancer-associated fibroblasts (CAFs) in head and neck cancer (HNC), thereby promoting tumorigenesis via modifying the tumor microenvironment. Herein, we have performed studies to investigate POSTN and its role in HNC microenvironment. Our results indicated that POSTN was significantly up-regulated in HNCs, especially in the tissues with lymph node metastasis. Moreover, POSTN was highly enriched in the stroma of cancer tissues and produced mainly by CAFs. More importantly, we have pinpointed TGF-β3 as the major upstream molecular that triggers the induction of POSTN in CAFs. As such, during the interaction between fibroblasts and cancer cells, the increased stromal POSTN induced by TGF-β3 directly accelerated the growth, migration and invasion of cancer cells. Hence, our study has provided a novel modulative role for POSTN on HNC progression and further reveals POSTN as an effective biomarker to predict metastasis as well as a potential cancer therapeutic target

Purification of QsdH and identification of AHL-degrading activity of QsdH.

<p>(A) Analysis of the expression and purification of QsdH protein by 12% SDS-PAGE. M is the standard molecular weight markers (TaKaRa); Lanes 1 and 2 are uninduced and induced cell lysates of <i>E. coli</i> BL21 (DE3) harboring pGEX-6p-<i>qsdH</i>, respectively. Lane 3 is the purified QsdH from <i>P. byunsanensis</i> 1A01261. The protein bands of GST-QsdH and QsdH are marked respectively. (B) AHL-degrading activity of purified QsdH. The solution of purified QsdH was mixed into the reaction buffer containing 5 µM 3OC8HSL (final concentration) and incubated at 40°C for 30 min. The residual 3OC8HSL was detected by <i>A. tumefaciens</i> strain NT1. The control consisted of 5 µM 3OC8HSL in reaction buffer incubated at 40°C for 30 min.</p

Effects of metal ions on QsdH activity for 3OC8HSL.

<p>Effects of metal ions on QsdH activity for 3OC8HSL.</p

Bacterial strains and plasmids used in the study.

<p>MCCC, Marine Culture Collection of China.</p

QsdH is localized in the inner membrane of a RND-type multidrug efflux transporter.

<p>The alignment of the ORF protein with two multidrug efflux pumps was showed. 1, ORF protein; 2, CzcA family heavy metal efflux protein from <i>Alteromonas</i> spp. SN2 (GI: 333892934); and 3, CzcA family heavy metal efflux protein from <i>Pseudoalteromonas atlantica</i> T6c (GI: 109898348). Structural predictions illustrate 12 transmembrane helices with two perisplasmic loops located between TM1 and TM2 and between TM7 and TM8 in the ORF protein (RND-type efflux transporter). The 12 transmembrane helices are shaded with a green box, and the typical signal peptide is marked in the ORF protein with a red box. Using <i>in silico</i> prediction, the GDSL hyrolase, QsdH, was predicted to form the first perisplasmic loop of the RND-type multidrug efflux transporter.</p

Screening an AHL-degrading activity gene <i>qsdH</i> and the physical map of <i>qsdH</i> locus.

<p>(A) The AHL-degrading activity of <i>E. coli</i> DH5α harboring different plasmids detected with biosensor <i>A. tumefaciens</i> strain NT1. CK, pUC118, used as a control; 1, pUC118-<i>sm20</i>, which was the screened positive transformant; 2, pUC118-<i>orf</i> (encodes the full ORF protein); 3, pUC118-<i>orf'</i> (encodes the ORF protein removing the N-terminal signal peptide); and 4, pUC118-<i>qsdH</i>. (B) A schematic representation of the complete ORF, which contains an N-terminal GDSL hydrolase domain and a RND-type multidrug efflux protein domain with the GDSL-lipolytic enzyme named QsdH.</p

Attenuation of potato pathogenicity of <i>Erwinia carotovora</i> by recombinant QsdH- producing <i>E. coli</i>.

<p>1, saline solution; 2, <i>E. coli</i> carrying pGEX-6p-1; 3, <i>E. coli</i> carrying pGEX-6p-<i>qsdH</i>; 4, <i>E. carotovora</i>; 5, mixture of <i>E. carotovora</i> and <i>E. coli</i> carrying pGEX-6p-1; 6, mixture of <i>E. carotovora</i> and <i>E. coli</i> carrying pGEX-6p-<i>qsdH</i>.</p