Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers.

Oral squamous cell carcinoma (OSCC) is a common public health problem worldwide with poor prognosis, which is largely due to lymph node metastasis and recurrence. Identification of specific molecular markers of OSCC with lymph node metastasis would be very important for early and specific diagnosis. In this study, we screened for the potential prognosis markers via unbiased transcriptomic microarray analysis in paired two OSCC cell lines, a lymph node metastatic HN12 cell line and a low metastatic parental HN4 cell line. The results showed that vimentin, with 87-fold increase of expression, was on the top of all upregulated genes in metastatic HN12 cells compared to non-metastatic HN4 cells. Treatment of non-metastatic HN4 cells with TGF-β1 induced epithelial to mesenchymal transition (EMT), with increased vimentin expression as well as enhanced migration activity. Consistently, knockdown of vimentin via siRNA resulted in suppressed invasion and migration activities of HN12 cells, suggesting an essential role of vimentin in EMT-related functions of OSCC cells. Finally, immunohistochemical (IHC) staining analysis showed that high vimentin expression was strongly associated with high lymph node metastases (p < 0.05), and poor overall survival (p < 0.05) in OSCC patients. Thus, high vimentin expression is strongly associated with increased metastatic potential, and may serve as a prediction marker for poor prognosis in OSCC patients.

Cancer cells that succeed in forming metastasis need to be reprogrammed to evade immune surveillance and survive in a new microenvironment. This is facilitated by metastatic niches that are either postformed through reciprocal signaling between tumor cells and local stromal cells or preformed as premetastatic niches before tumor cell arrival. IL6/STAT3 signaling is aberrantly activated in lung tumorigenesis and metastasis, however, the roles and mechanisms of action of IL6 remain controversial. Here, we showed that blockade of intrinsic STAT3 signaling in lung tumor cells suppressed lung metastasis in immune-competent syngeneic mice, but not in immune-deficient nude mice. Consistently, repression of STAT3 signaling in tumor cells made them susceptible to T-cell-mediated cytotoxicity. Thus, STAT3mediated immunosuppression is crucial for metastasis. Noticeably, lung metastasis was greatly increased in Gprc5a-knockout (ko; 5a À/À ) mice compared with wild-type mice, which correlated with upre-gulated IL6 in the tumor microenvironment. Depletion of IL6 via combined deletion of Il6 and Gprc5a genes almost completely eliminated lung metastasis in Gprc5a-ko/Il6-ko (5a À/À ;Il6 À/À ) mice. Mechanistically, dysregulated IL6 reprogrammed the STAT3 pathway in metastatic tumor cells, and induced recruitment of myeloid-derived suppressor cells and polarized macrophages to evade host immunity. Consistently, IHC staining showed that activated STAT3 correlated with repressed infiltration of CD8 þ T cells in non-small cell lung cancer. Therefore, IL6/STAT3 signaling is crucial for orchestrating premetastatic niche formation and immunosuppression in lung.Significance: IL6 plays important roles not only in cell autonomous propensity for metastasis, but also in establishing the metastatic niche.

Chronic inflammation has been linked to promotion of tumorigenesis and metastasis in lung. However, due to lack of a relevant animal model for characterization, the underlying mechanism remains elusive. Lung tumor suppressor gene Gprc5a-knockout (ko) mice are susceptible to lung inflammation, tumorigenesis and metastasis, which resembles the pathological features in human patients. Here, we showed that PTGES/PGE 2 signaling was highly associated with lung tumorigenesis and metastasis in Gprc5a-ko mice. Interestingly, Ptges-knockout in mouse lung tumor cells, although reduced their stemness and EMT-like features, still formed tumors and lung metastasis in immune-deficient nude mice, but not in immune-competent mice. This suggests that the major role of PTGES/PGE 2 signaling in tumorigenicity and lung metastasis is through immunosuppression. Mechanistically, PTGES/PGE 2 signaling intrinsically endows tumor cells resistant to T-cell cytotoxicity, and induces cytokines extrinsically for MDSC recruitment, which is crucial for suppression of T-cell immunity. Importantly, targeting PGE 2 signaling in Gprc5a-ko mice by PTGES inhibitor suppressed MDSC recruitment, restored T cells, and significantly repressed lung metastasis. Thus, PTGES/PGE 2 signaling links immunosuppression and metastasis in an inflammatory lung microenvironment of Gprc5a-ko mouse model.