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While initiation is established as a critical step in tumorigenesis, the identity of the cell of origin for lung adenocarcinoma and the mechanism controlling susceptibility to initiation remain elusive. Here we show that lung tumor suppressor Gprc5a-knockout (KO) mice are susceptible to initiation of lung tumorigenesis. Bronchioalveolar stem cells (BASC) and alveolar type 2 (AT2) cells were aberrantly expanded in Gprc5a-KO mouse lungs compared with those in wild-type (WT) mice, suggesting that Gprc5a-KO might confer susceptibility to initiation by increasing the cell of origin in mouse lungs. BASCs from Gprc5a-KO mice (KO-BASC) exhibited significantly increased stemness and self-renewal potential and reduced differentiation capacity compared with BASCs from WT mice (WT-BASC). AT2 cells did not possess self-renewal potential regardless of Gprc5a status. KO-BASCs expressed a stem-like gene profile with upregulated Abcg2, EGFR, and NF-κB signaling compared with WT-BASCs. Blockade of EGFR and NF-κB signaling inhibited both expansion of BASC and AT2 cells and lung tumorigenesis. Abcg2 was expressed in active KO-BASCs as well as in lung tumor cells but not in quiescent WT-BASCs or AT2 cells, supporting that lung adenocarcinoma cells are derived from Abcg2-positive KO-BASCs (active). Taken together, Gprc5a deletion leads to expansion of active BASCs via dysregulated EGFR and NF-κB signaling that confers susceptibility to initiation of lung tumorigenesis, marking Abcg2-positive BASCs as candidate cell of origin for lung adenocarcinoma. Significance: Identification of active bronchioalveolar stem cells as lung adenocarcinoma cells of origin provides insights into mechanisms of lung tumorigenesis and could facilitate development of effective strategies for cancer prevention and therapy. See related commentary by Osborne and Minna, p. 972

Supplementary Data from Identification of Active Bronchioalveolar Stem Cells as the Cell of Origin in Lung Adenocarcinoma

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, STAT3-mediated immunosuppression is crucial for metastasis. Noticeably, lung metastasis was greatly increased in Gprc5a-knockout (ko; 5a−/−) mice compared with wild-type mice, which correlated with upregulated 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−/−;Il6−/−) 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+ 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.

While initiation is established as a critical step in tumorigenesis, the identity of the cell of origin for lung adenocarcinoma and the mechanism controlling susceptibility to initiation remain elusive. Here we show that lung tumor suppressor Gprc5a-knockout (KO) mice are susceptible to initiation of lung tumorigenesis. Bronchioalveolar stem cells (BASC) and alveolar type 2 (AT2) cells were aberrantly expanded in Gprc5a-KO mouse lungs compared with those in wild-type (WT) mice, suggesting that Gprc5a-KO might confer susceptibility to initiation by increasing the cell of origin in mouse lungs. BASCs from Gprc5a-KO mice (KO-BASC) exhibited significantly increased stemness and self-renewal potential and reduced differentiation capacity compared with BASCs from WT mice (WT-BASC). AT2 cells did not possess self-renewal potential regardless of Gprc5a status. KO-BASCs expressed a stem-like gene profile with upregulated Abcg2, EGFR, and NF-κB signaling compared with WT-BASCs. Blockade of EGFR and NF-κB signaling inhibited both expansion of BASC and AT2 cells and lung tumorigenesis. Abcg2 was expressed in active KO-BASCs as well as in lung tumor cells but not in quiescent WT-BASCs or AT2 cells, supporting that lung adenocarcinoma cells are derived from Abcg2-positive KO-BASCs (active). Taken together, Gprc5a deletion leads to expansion of active BASCs via dysregulated EGFR and NF-κB signaling that confers susceptibility to initiation of lung tumorigenesis, marking Abcg2-positive BASCs as candidate cell of origin for lung adenocarcinoma.Significance:Identification of active bronchioalveolar stem cells as lung adenocarcinoma cells of origin provides insights into mechanisms of lung tumorigenesis and could facilitate development of effective strategies for cancer prevention and therapy.See related commentary by Osborne and Minna, p. 972

Supplementary Fig. S1-S6, supplementary methods, supplementary table S1-S2. Supplementary Table S1. Primers used in the study; Supplementary Table S2. Antibodies information for flow cytometry analysis; Figure S1. IL6/ Gp130 pathway is involved in maintenance of stemness and in suppression of host immunity for lung metastasis; Figure S2. IL6 signaling is essential for lung metastasis in Gprc5a-ko mice lung tissues; Figure S3. IL6 signaling is essential for Lewis lung carcinoma cells in lung metastasis in Gprc5a-ko mice lung tissues; Figure S4. IL-6 signaling mediates the inhibition on innate immunity in Gprc5a-ko mouse lungs; Figure S5. IL-6 signaling orchestrates the inhibition on innate and adaptive immunity in Gprc5a-ko mouse lungs; Figure S6. Proposed model of IL-6/STAT3 signaling in lung metastasis

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Supplementary Figures S1-S8. HEK293T cells were co-transfected with the plasmids of GPRC5A-myc, or myc-tagged GPRC5A mutants (∆C, ∆N, ∆7TM, and 7TM) as indicated ; Immunoblot was performed for p-EGFR (Y1068) and p-STAT3 (Y705) proteins from mouse lung tissues with the treatment as indicated ; IHC Staining for EGFR in the lung tissues of KO-NNK-12m (NNK-treated Gprc5a-/-mouse at age of 12 month) ; Representative pictures of IHC Staining for EGFR in two tumors (#282-1 and #282-2) identified in the same Gprc5a-/-mouse lung (#282) that was treated with NNK at age of 2 month and was sacrificed at age of 12 months (Gprc5a-ko-NNK-12m) ; Images of IHC Staining with normal rabbit IgG (upper) and anti-EGFR (bottom) in the tumor tissues (left) and the epithelium of small and terminal bronchioles (S/TB) of the lung from Gprc5a-ko mice treated with NNK at age of 12 months ; IHC Staining for p-STAT3 (Y705) in the lung tissues of KO-NNK-12m (NNK-treated Gprc5a-/- mouse at age of 12 month) ; Image of IHC Staining for Ki67 in the lung tissue of Gprc5a-ko-NNK-12m mouse which contains both the normal epithelium of small and terminal bronchioles (S/TB) (upper left) or tumor (bottom right) ; GPRC5A locus is located in the 12p12.3-p13 region which is frequently lost human NSCLC .

Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulations play a critical role. Lung tumor suppressor GPRC5A is repressed in most non-small cell lung cancer (NSCLC), however the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induces GPRC5A repression both in vitro and in vivo. Intriguingly, trans-activation of NF-κB downstream targets is not required, but the trans-activation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in GPRC5A promoter. Instead, p65 was complexed with RARα/β, and recruited to the RA-response element (RARE) site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complex, and suppressed transcription. Noticeably, phosphorylation on Serine276 of p65 is required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression is through suppression of histone H3K9ac, but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a HDAC inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via complex with RARα/β and mediates epigenetic repression via suppression of H3K9ac.

Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc−. Xc− is a sodium-independent antiporter passively driven by concentration gradients from extracellular cystine and intracellular glutamate across the cell membrane. Increased uptake of cystine via Xc− in cancer cells increases the level of extracellular glutamate, which would subsequently restrain cystine uptake via Xc−. Cancer cells must therefore evolve a mechanism to overcome this negative feedback regulation. In this study, we report that glutamate transporters, in particular SLC1A1, are tightly intertwined with cystine uptake and GSH biosynthesis in lung cancer cells. Dysregulated SLC1A1, a sodium-dependent glutamate carrier, actively recycled extracellular glutamate into cells, which enhanced the efficiency of cystine uptake via Xc− and GSH biosynthesis as measured by stable isotope-assisted metabolomics. Conversely, depletion of glutamate transporter SLC1A1 increased extracellular glutamate, which inhibited cystine uptake, blocked GSH synthesis, and induced oxidative stress-mediated cell death or growth inhibition. Moreover, glutamate transporters were frequently upregulated in tissue samples of patients with non–small cell lung cancer. Taken together, active uptake of glutamate via SLC1A1 propels cystine uptake via Xc− for GSH biosynthesis in lung tumorigenesis. Significance: Cellular GSH in cancer cells is not only determined by upregulated Xc− but also by dysregulated glutamate transporters, which provide additional targets for therapeutic intervention.

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, STAT3-mediated immunosuppression is crucial for metastasis. Noticeably, lung metastasis was greatly increased in Gprc5a-knockout (ko; 5a−/−) mice compared with wild-type mice, which correlated with upregulated 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−/−;Il6−/−) 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+ 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/PGE2 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/PGE2 signaling in tumorigenicity and lung metastasis is through immunosuppression. Mechanistically, PTGES/PGE2 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 PGE2 signaling in Gprc5a-ko mice by PTGES inhibitor suppressed MDSC recruitment, restored T cells, and significantly repressed lung metastasis. Thus, PTGES/PGE2 signaling links immunosuppression and metastasis in an inflammatory lung microenvironment of Gprc5a-ko mouse model.

GPRC5A, a retinoic acid induced gene, is preferentially expressed in lung tissue. Gprc5a gene deletion leads to spontaneous lung tumor development. However, the mechanism of Gprc5a-mediated lung tumor suppression is not fully understood. Here we showed that MDM2, a p53-negative regulator, was dysregulated in Gprc5a-knockout (ko) mouse tracheal epithelial cells (KO-MTEC) compared to wild type ones. Targeting MDM2 in 1601-a Gprc5a-ko mouse derived lung tumor cell line-and A549-human lung cancer cells, by MDM2 inhibitor Nutlin-3a or small hairpin RNA (sh-RNA)-restored p53 signaling pathway, reduced cancer stem cell markers, and inhibited tumorigenicity. This suggests that dysregulated MDM2 pathway is essential for the oncogenic activities of these cells. MDM2 was found to be stabilized mainly by activated EGFR signaling as targeting EGFR by Erlotinib or sh-RNA repressed MDM2 in a transcription-independent manner. Importantly, overexpression of MDM2 and reduced GPRC5A expression at both protein and mRNA levels were frequently found in clinical human lung cancer tissues. Taken together, GPRC5A deficiency contributes to dysregulated MDM2 via activated EGFR signaling, which promotes lung tumor development.

Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.

Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc

Lung squamous cell carcinomas (SCCs) are highly aggressive tumors, and there is currently no effective targeted therapy owing to the lack of specific mutation targets. Compared with lung adenocarcinoma (ADCs), lung SCCs reportedly utilized higher levels of glucose metabolism to meet the anabolic and catabolic needs required to sustain rapid tumor growth. Hexokinase 2 (HK2) is an enzyme that catalyzes the rate-limit and first committed step in glucose metabolism. Here, we investigated the expression and effect of HK2 in lung SCCs. We found a significantly higher HK2 expression in lung SCCs, but not lung ADC or normal tissues. HK2 depletion or inhibition decreased the glycolysis and tumor growth via activating AMPK signaling pathway, which downregulated mTORC1 activity. Furthermore, we found an increased oxygen respiration rate compensating for HK2 depletion. Thus, metformin treatment showed combinatorial therapeutic value, which resulted in greater induction of lung SCC apoptosis in vitro and in vivo. Our study suggests that HK2 depletion in combination with metformin might be a novel effective strategy for lung SCCs therapy.

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/PGE2 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/PGE2 signaling in tumorigenicity and lung metastasis is through immunosuppression. Mechanistically, PTGES/PGE2 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 PGE2 signaling in Gprc5a-ko mice by PTGES inhibitor suppressed MDSC recruitment, restored T cells, and significantly repressed lung metastasis. Thus, PTGES/PGE2 signaling links immunosuppression and metastasis in an inflammatory lung microenvironment of Gprc5a-ko mouse model.

Early metastasis and local recurrence are the major causes of mortality and poor prognosis of non-small cell lung cancer (NSCLC). However, the underlying mechanisms of these processes are poorly understood. In this study, we aimed to investigate the roles of the PTGES/PGE(2) pathway in lung cancer progression. We found that prostaglandin E synthase (PTGES), a key enzyme for PGE(2) synthesis in the arachidonic acid pathway, was highly dysregulated in NSCLC. Dysregulated PTGES was essential for the promotion of tumor migration and metastasis of NSCLC cells. Knockdown of PTGES in lung cancer cells resulted in suppressed cell migration, which was reversed by exogenous PGE(2). Consistent with this, PTGES knockdown also reduced the expression of CSC markers, tumor sphere formation, colony forming activity, tumorigenicity, and lung metastasis in vivo. Dysregulated PTGES is mainly attributed to protein stabilization by USP9X, a deubiquitination enzyme. USP9X physically interacted with PTGES and prevented it from proteasome-directed degradation via deubiquitination. Consistent with this, USP9X expression was highly correlated with PTGES expression in NSCLC tumor tissues. Taken together, our results show that the upregulated USP9X-PTGES-PGE(2) axis contributes significantly to the metastatic features of NSCLC.

Iron dysregulation is associated with several diseases, including lung cancer, but the underlying mechanism is yet unknown. Iron directly binds CDK1, which is upregulated in several cancers, thereby promoting JAK1 phosphorylation and activation of STAT3 signaling to promote colorectal carcinogenesis. This study aimed to investigate the role of iron/CDK1/STAT3 signaling in lung carcinogenesis. We found that iron-dependent CDK1 activity upregulated IL-6 receptor subunit GP130 post-transcriptionally via phosphorylation of 4E-BP1, which is critical for activation of JAK/STAT3 signaling. CDK1 and STAT3 are essential for iron-mediated colony formation in lung cancer cell lines. CDK1 knockdown and iron chelator DFO decreased tumorigenicity and GP130/STAT3 signaling in vivo. Moreover, CDK1/GP130/STAT3 signaling were elevated in lung cancer tissues compared with adjacent normal lung tissues. Altogether, the present results suggest that CDK1 inhibition and iron deprivation are potential strategies to target GP130/STAT3 signaling to suppress lung cancer.

Aims Lung cancer remains the leading cause of cancer incidence and mortality. Although cigarette smoke is regarded as a high risk factor for lung tumor initiation, the role of the lung tumor suppressor GPRC5A in smoking-induced lung cancer is unclear. Main methods We obtained two lung cancer cohorts from the TCGA and GEO databases. Bioinformatics analysis showed differential gene expression in the cohorts. Quantitative real-time PCR, Western Blot and Gprc5a−/− mice uncovered the relationship between cigarette smoke and lung cancer in the GPRC5A deletion system in vitro and in vivo. Key findings Bioinformatics analysis showed that the smoking lung cancer patients with low expression of GPRC5A had poor overall survival compared to the patients with high GPRC5A expression. Further analysis revealed that cancer-related stemness pathways such as the Hippo signaling pathway were induced in smoking patients with low GPRC5A expression. Additionally, we detected enriched expression of WNT5A and DLX5 in normal human lung epithelial 16HBE cells and human lung cancer H1299 cells in vitro. A relationship between cigarette smoke extract (NNK) and lung tumor initiation was observed in Gprc5a−/− mice. Significance The lung tumor suppressor gene GPRC5A played a protective role in cigarette smoke-induced lung tumor initiation, providing a target for the prevention of lung cancer development and monitoring of prognosis.

Induction of cancer stem cell (CSC) characters and epithelial mesenchymal transition (EMT) features are crucial in tumor initiation, progression and metastasis. However, underlying mechanisms remain incompletely understood. Here, we showed that ENPP1 plays an important role in inducing and maintaining EMT phenotypes and CSC features in lung cancer. ENPP1 is upregulated in lung cancer cells. ENPP1-knockdown in lung cancer HCC827 cells and A549 cells resulted in suppressed colonogenic formation, anchorage-independent growth in vitro, and tumorigenicity in vivo. ENPP1-knockdown also reduced expression of CSC makers, including ABCG2, SOX2, NANOG, and CD44. Moreover, ENPP1-knockdown reversed TGFβ-induced EMT phenotypes, including cell migration, E-cadherin repression and vimentin induction. Finally, upregulated ENPP1 was identified in majority of human lung tumor tissues compared to adjacent normal lung tissues. Taken together, our study demonstrates that dysregulated ENPP1 contributes to increased malignancy of human lung cancer by inducing CSC-features, and EMT-like phenotypes.

Exposure to crystalline silica is suggested to increase the risk for a variety of lung diseases, including fibrosis and lung cancer. However, epidemiological evidences for the exposure-risk relationship are ambiguous and conflicting, and experimental study from a reliable animal model to explore the relationship is lacking. We reasoned that a mouse model that is sensitive to both lung injury and tumorigenesis would be appropriate to evaluate the exposure-risk relationship. Previously, we showed that, Gprc5a-/- mice are susceptible to both lung tumorigenesis and endotoxin-induced acute lung injury. In this study, we investigated the biological consequences in Gprc5a-/- mouse model following silica exposure. Intra-tracheal administration of fine silica particles in Gprc5a-/- mice resulted in more severe lung injury and pulmonary inflammation than in wild-type mice. Moreover, an enhanced fibrogenic response, including EMT-like characteristics, was induced in the lungs of Gprc5a-/- mice compared to those from wild-type ones. Importantly, increased hyperplasia or neoplasia coincided with silica-induced tissue injury and fibrogenic response in lungs from Gprc5a-/- mice. Consistently, expression of MMP9, TGFβ1 and EGFR was significantly increased in lungs from silica-treated Gprc5a-/- mice compared to those untreated or wild-type ones. These results suggest that, the process of tissue repair coincides with tissue damages; whereas persistent tissue damages leads to abnormal repair or neoplasia. Thus, silica-induced pulmonary inflammation and injury contribute to increased neoplasia development in lungs from Gprc5a-/- mouse model.

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.

Pyrrolidine dithiocarbamate (PDTC) is widely used as an antioxidant or an NF-κB inhibitor. It has been reported to inhibit the replication of human rhinoviruses, poliovirus, coxsackievirus, and influenza virus. In this paper, we report that PDTC could inhibit the replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2). PDTC suppressed the expression of HSV-1 and HSV-2 viral immediate early (IE) and late (membrane protein gD) genes and the production of viral progeny. This antiviral property was mediated by the dithiocarbamate moiety of PDTC and required the presence of Zn 2+ . Although PDTC could potently block reactive oxygen species (ROS) generation, it was found that this property did not contribute to its anti-HSV activity. PDTC showed no activity in disrupting the mitogen-activated protein kinase (MAPK) pathway activation induced by viral infection that was vital for the virus's propagation. We found that PDTC modulated cellular ubiquitination and, furthermore, influenced HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced PML stability in the nucleus, resulting in the inhibition of viral gene expression. These results suggested that the antiviral activity of PDTC might be mediated by its dysregulation of the cellular ubiquitin-proteasome system (UPS).

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

// Beibei Sun 1, * , Wenzheng Guo 2, 3, * , Song Hu 1 , Feng Yao 4 , Keke Yu 5 , Jie Xing 5 , Ronghua Wang 1 , Hongyong Song 2, 3 , Yueling Liao 2, 3 , Tong Wang 2, 3 , Pengfei Jiang 6 , Baohui Han 1, 7 , Jiong Deng 1, 2, 3 1 Translation Medicine Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China 2 Key Laboratory of Cell Differentiation and Apoptosis of Chinese Minister of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China 3 Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, China 4 Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China 5 Department of Biobank, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China 6 Department of Laboratory Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China 7 Department of Pulmonary Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China * These authors contributed equally to this work Correspondence to: Baohui Han, email: xkyyhan@gmail.com Jiong Deng, email: jiongdeng@shsmu.edu.cn Keywords: lung cancer, biomarker, ceruloplasmin, Gprc5a Received: November 15, 2016 Accepted: December 31, 2016 Published: January 10, 2017 ABSTRACT Lung cancer is the leading cause of cancer death. As most of lung cancer patients were diagnosed with the advanced stage, early detection is considered as the most effective strategy to reduce high mortality. Thus, it is desirable to identify specific biomarkers at early stages of lung tumorigenesis. GPRC5A is a lung tumor suppressor gene. GPRC5A deficiency is linked to lung cancer development. We hyposthesized that, dysregulated gene expression that results from Gprc5a deficiency may provide potential biomarkers at early stages of lung tumorigenesis. By analysis of top 20 upregulated genes in mouse tracheal epithelial cells (MTEC) of Gprc5a knockout (KO) vs wild-type (WT), we found that ceruloplasmin, lipocalin-2, and periostin are not only upregulated in lung epithelial cells of Gprc5a-ko mice, but also expressed at high levels in lung tumor tissues of Gprc5a-ko mice. This suggests that increased expression of these genes is associated with lung tumorigenesis. Importantly, expression of ceruloplasmin, lipocalin-2, and periostin has also been found to be significantly increased, both at mRNA and protein levels, in the lung tissues from NSCLC patients, which is correlated with repressed GPRC5A. Thus, dysregulated ceruloplasmin, lipocalin-2, and periostin may be used as potential biomarkers at early stages of lung tumorigenesis.

Salivary adenoid cystic carcinoma (SACC) is characterized by invasive local growth and a high incidence of lung metastasis. Patients with lung metastasis have a poor prognosis. Treatment of metastatic SACC has been unsuccessful, largely due to a lack of specific targets for the metastatic cells. In this study, we showed that epidermal growth factor receptors (EGFR) were constitutively activated in metastatic lung subtypes of SACC cells, and that this activation was induced by autocrine expression of epiregulin (EREG), a ligand of EGFR. Autocrine EREG expression was increased in metastatic SACC-LM cells compared to that in non-metastatic parental SACC cells. Importantly, EREG-neutralizing antibody, but not normal IgG, blocked the autocrine EREG-induced EGFR phosphorylation and the migration of SACC cells, suggesting that EREG-induced EGFR activation is essential for induction of cell migration and invasion by SACC cells. Moreover, EREG-activated EGFR stabilized Snail and Slug, which promoted EMT and metastatic features in SACC cells. Of note, targeting EGFR with inhibitors significantly suppressed both the motility of SACC cells in vitro and lung metastasis in vivo. Finally, elevated EREG expression showed a strong correlation with poor prognosis in head and neck cancer. Thus, targeting the EREG-EGFR-Snail/Slug axis represents a novel strategy for the treatment of metastatic SACC even no genetic EGFR mutation.

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.

GPRC5A is a G-protein–coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a−/− mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a−/− mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a−/− MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a−/− mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non–small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801–14. ©2015 AACR.

Background Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are among the most prevalent human pathogens in both industrialised and developing regions. In the current study, we reported that two zinc ionephores, pyrrolidine dithiocarbamate (PDTC) and pyrithione (PT) could inhibit herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) replication. Methods Viral replication was evaluated via detecting HSV late gene product (Glycoprotein, gD) using In-cell Western. NF-κB, MAPK activation, viral immediate-early genes expression and profile of cellular ubiquitin-conjugates were determined by Western blot. Protein localization was investigated via immunofluorescence staining. Viral protein expression on mRNA level was quantified by realtime-PCR. 26S Proteasome activation was determined using fluorogenic substrate, Suc-LLVY-AMC. Results PDTC and PT inhibited HSV-1 and HSV-2 gD expression and the production of viral progeny, which was dependent on zinc ion. Further studies showed that these two compounds suppressed the HSV immediate-early gene, the infected cell polypeptide 4 (ICP4) expression, but had less effect on ICP0. HSV infection could interact with cellular ubiquitin-proteasome system (UPS) and cause loss of high molecular weight ubiquitin-conjugates. It was found that PDTC and PT could interfere UPS, leading to the inhibition of HSV-2-induced IκB-α degradation to prevent NF-κB activation and enhanced PML stability in nucleus. However, PDTC and PT did not show direct inhibition of 26S proteasome activity. Instead, these two zinc ionphores induced import of extracellular zinc ions into cells, which facilitated dysregulation of proteasome function and accumulation of intracellular ubiquitin-conjugates. Other evidence was that the inhibitors of ubiquitin activating enzyme E1 and deubiquitinase also inhibited HSV replication, implying that UPS was required for effective replication of HSV-1 and HSV-2 Conclusion Homostasis of ubiquitin cycle and UPS were critical for HSV gene expression and replication and that the ubiquitin-proteasome pathway is a potential drug target for HSV infection.

Background G protein–coupled receptor family C group 5 member A (GPRC5A), a retinoic acid-inducible gene, is a lung tumor suppressor. Previously, we showed that repression of GPRC5A expression was associated with pathologic differentiation grade of oral squamous cell carcinomas (OSCC) and overexpression of GPRC5A gene inhibited the malignant phenotype in OSCC cells, suggesting that GPRC5A also functions as a tumor suppressor in oral cancer. However, the molecular mechanisms underlying GPRC5A deficiency in head and neck squamous cell carcinoma (HNSCC) are still unclear. Methods In this study, we used Western blot analysis and immunohistochemical (IHC) staining to investigate the expression of GPRC5A in both HNSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental HNSCC cells were characterized for their biological activities in anchorage-independent growth. Results IHC analysis showed that, GPRC5A expression was high in normal tissue, but gradually decreased in oral leukoplakia, a precancerous stage, and greatly suppressed in primary cancer. Repression of GPRC5A was correlated with activated STAT3, which associates with aggressive clinicopathological features in HNSCC patients. Moreover, overexpression of GPRC5A suppressed IL-6-induced-STAT3 activation and inhibited anchorage-independent growth in HNSCC cells. Conclusions Repressed GPRC5A associates with increased tumor grade and activated STAT3, which may be used as a prognostic marker for tumor progression of HNSCC.