Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Lipolysis-stimulated lipoprotein receptor promote lipid uptake and fatty acid oxidation in gastric cancer.

BACKGROUND: Lipolysis-stimulated lipoprotein receptor (LSR), a lipid receptor, is associated with cancer progression. However, detailed effects on intracellular metabolism are unclear. We aimed to elucidate the mechanism of LSR-mediated lipid metabolism in gastric cancer. METHODS: We investigated lipid metabolic changes induced by lipoprotein administration in gastric cancer cells and evaluated the significance of LSR expression and lipid droplets formation in gastric cancer patients. The efficacy of inhibiting ß-oxidation in gastric cancer cells was also examined in vitro and vivo. RESULTS: In gastric cancer cells, LSR promoted cellular uptake of lipoprotein and cell proliferation. Furthermore, the inhibition of LSR in gastric cancer cells expressing high levels of LSR counteracted both effects. Immunohistochemical analysis of human gastric cancer tissues showed that the increase in lipid droplets via LSR is a factor that influences prognosis. Lipidomics analysis of LSR-high-expressing gastric cancer cells revealed an increase in ß-oxidation. Based on these results, we used etomoxir, a ß-oxidation inhibitor, and found that it inhibited cell proliferation as well as the suppression of LSR. Similarly, in a mouse xenograft model of LSR-highly expressing gastric cancer cells, the tumor growth effect of high-fat diet feeding was counteracted by etomoxir, consistent with the Ki-67 labeling index. CONCLUSIONS: We demonstrated that lipids are taken up into gastric cancer cells via LSR and cause an increase in ß-oxidation, resulting in the promotion of cancer progression. Controlling LSR-mediated lipid metabolism may be a novel therapeutic strategy for gastric cancer.

Combination of pimitespib (TAS-116) with sunitinib is an effective therapy for imatinib-resistant gastrointestinal stromal tumors.

Despite the effectiveness of imatinib, most gastrointestinal stromal tumors (GISTs) develop resistance to the treatment, mainly due to the reactivation of KIT tyrosine kinase activity. Sunitinib, which inhibits the phosphorylation of KIT and vascular endothelial growth factor (VEGF) receptor, has been established as second-line therapy for GISTs. The recently-developed heat shock protein 90 (HSP90) inhibitor pimitespib (PIM; TAS-116) demonstrated clinical benefits in some clinical trials; however, the effects were limited. The aim of our study was therefore to clarify the effectiveness and mechanism of the combination of PIM with sunitinib for imatinib-resistant GISTs. We evaluated the efficacy and mechanism of the combination of PIM with sunitinib against imatinib-resistant GIST using imatinib-resistant GIST cell lines and murine xenograft models. In vitro analysis demonstrated that PIM and sunitinib combination therapy strongly inhibited growth and induced apoptosis in imatinib-resistant GIST cell lines by inhibiting KIT signaling and decreasing auto-phosphorylated KIT in the Golgi apparatus. In addition, PIM and sunitinib combination therapy enhanced antitumor responses in the murine xenograft models compared to individual therapies. Further analysis of the xenograft models showed that the combination therapy not only downregulated the KIT signaling pathway but also decreased the tumor microvessel density. Furthermore, we found that PIM suppressed VEGF expression in GIST cells by suppressing protein kinase D2 and hypoxia-inducible factor-1 alpha, which are both HSP90 client proteins. In conclusion, the combination of PIM and sunitinib is effective against imatinib-resistant GIST via the downregulation of KIT signaling and angiogenic signaling pathways.

The Skin-Liver Axis Modulates the Psoriasiform Phenotype and Involves Leucine-Rich α-2 Glycoprotein.

Leucine-rich α-2 glycoprotein (LRG), one of the acute phase proteins mainly produced by the liver, similar to C-reactive protein, has been recognized as an inflammatory biomarker for rheumatoid arthritis and inflammatory bowel diseases. We recently demonstrated that LRG was also increased in the sera of psoriasis patients and correlated well with disease activity with a sensitivity and specificity much higher than C-reactive protein; however, whether LRG mechanistically contributed to the pathogenesis of psoriasis remained unclear. In this study, we explored the role of LRG in psoriasiform inflammation using LRG-knockout (KO) mice in an imiquimod (IMQ)-mediated model. Following topical treatment with IMQ, serum levels of LRG and its expression in the liver were abruptly elevated. Similarly, an acute surge of proinflammatory cytokines was observed in the liver, including IL-1ß, TNF-α, and IL-6, although LRG-KO mice showed delayed responses. LRG-KO mice showed less skin inflammation in the IMQ model than wild-type mice. K5.Stat3C mice developed psoriasis-like lesions following tape stripping, which also abruptly induced LRG expression in the liver. A deficiency of Lrg mitigated tape stripping-induced lesions, similar to the IMQ model. These results indicate that LRG modulates both feed-forward and feedback loops of cytokines in the skin-liver axis involved with psoriasiform inflammation.

Gene therapy with SOCS1 induces potent preclinical antitumor activities in oral squamous cell carcinoma.

BACKGROUND: Constitutive activation of STAT3 promotes oncogenesis and growth of oral squamous cell carcinoma (OSCC). We investigated the mechanism of action of suppressor of cytokine signaling 1 (SOCS1), an endogenous inhibitor of JAK, as gene therapy for OSCC. METHODS: Antitumor effect of SOCS1 was compared to JAK inhibitor I by cell proliferation assay, cell cycle analysis, and apoptosis analysis in vitro. In addition, antitumor effect was evaluated using xenograft mouse models in vivo. RESULTS: JAK inhibitor I inhibited the proliferation of KOSC2 cl3-43 or T3M-1 clone2 OSCC cell lines in vitro. While JAK inhibitor I arrested both cell lines at the G2/M phase, induction of apoptosis was observed in T3M-1 clone2 cells, but not KOSC2-cl3-43 cells. An adenoviral vector expressing SOCS1 (AdSOCS1) significantly decreased the proliferation of both OSCC cell lines and induced G2/M phase cell cycle arrest and apoptosis, suggesting that induction of apoptosis of KOSC2 cl3-43 cells by AdSOCS1 is regulated by the JAK/STAT independent pathway. Overexpression of SOCS1 inhibited activation of the JAK/STAT and p44/42 MAPK pathways, while JAK inhibitor I inhibited activation of the JAK/STAT pathway only. Consistently, expression of Mcl-1 was decreased by overexpression of SOCS1, but not JAK inhibitor I. Additionally, KOSC2 cl3-43 or T3M-1 clone2 OSCC cells were subcutaneously implanted in the flanks of two xenograft mouse models. As compared to a control adenovirus vector (AdLacZ), intratumor injection of AdSOCS1 significantly decreased the tumor volume and induced apoptosis in vivo. CONCLUSION: SOCS1 gene therapy may be a beneficial approach for the treatment of OSCC.

Targeted therapy for drug-tolerant persister cells after imatinib treatment for gastrointestinal stromal tumours.

BACKGROUND: Despite the effectiveness of tyrosine kinase inhibitors (TKI), gastrointestinal stromal tumours (GIST) develop after the withdrawal of TKI. Based on previous studies, a subpopulation of drug-tolerant cells called "persister cells" may be responsible for the recurrence and have thus, gained attention as a novel target in cancer therapy. METHODS: The metabolic changes were investigated in imatinib-derived persister GIST cells. We investigated the efficacy and the mechanism of GPX4 inhibitor, which is known as a major inducer of "ferroptosis". We also evaluated the effects of RSL3 to the gefitinib-derived persister lung cancer cells. RESULTS: We demonstrated a downregulation of glucose metabolism, subsequent decrease in the glutathione level and sensitivity to glutathione peroxidase 4 (GPX4) inhibitor, RSL3 in persister cells. As the cell death induced by RSL3 was found to be "iron-dependent" and "caspase-independent", loss of GPX4 function could have possibly induced selective persister cell ferroptotic death. In the xenograft model, we confirmed the inhibition of tumour regrowth after discontinuation of imatinib treatment. Moreover, RSL3 prevented the growth of gefitinib-derived persister lung cancer cells. CONCLUSIONS: RSL3 combined with TKI may be a promising therapy for both GIST and epidermal growth factor receptor-mutated lung cancer.

LSR promotes epithelial ovarian cancer cell survival under energy stress through the LKB1-AMPK pathway.

Lipolysis-stimulated lipoprotein receptor (LSR), also known as a component of tricellular tight junctions, is highly expressing in epithelial ovarian cancer (EOC). However, the biological role of LSR in EOC cells remains unclear. In this study, we evaluated liver kinase B1 (LKB1) mediated AMP-activated protein kinase (AMPK) activity and investigated the effect of LSR on EOC cell survival under energy stress. LSR increased the levels of phospho-AMPKα at Thr172 and phospho-acetyl-CoA carboxylase (ACC) at Ser79 via LKB1-AMPK pathway in glucose deprivation in vitro. The increase of P-AMPKα (Thr172) and P-ACC (Ser79) was also detected in tumor microenvironment in vivo. Meanwhile, LSR promoted LKB1 localization at the cell membrane of EOC cells. By cell survival analysis, LSR attenuated glucose deprivation-induced cell death in EOC cells in vitro. Our results suggest that LSR promotes EOC cell survival and tumor growth through the LKB1-AMPK pathway.

Propranolol suppresses gastric cancer cell growth by regulating proliferation and apoptosis.

BACKGROUND: Despite improvements in gastric cancer treatment, the mortality associated with advanced gastric cancer is still high. The activation of ß-adrenergic receptors by stress has been shown to accelerate the progression of several cancers. Accordingly, increasing evidence suggests that the blockade of ß-adrenergic signaling can inhibit tumor growth. However, the effect of ß-blockers, which target several signaling pathways, on gastric cancer remains to be elucidated. This study aimed to investigate the anti-tumor effects of propranolol, a non-selective ß-blocker, on gastric cancer. METHODS: We explored the effect of propranolol on the MKN45 and NUGC3 gastric cancer cell lines. Its efficacy and the mechanism by which it exerts anti-tumor effects were examined using several assays (e.g., cell proliferation, cell cycle, apoptosis, and wound healing) and a xenograft mouse model. RESULTS: We found that propranolol inhibited tumor growth and induced G1-phase cell cycle arrest and apoptosis in both cell lines. Propranolol also decreased the expression of phosphorylated CREB-ATF and MEK-ERK pathways; suppressed the expression of matrix metalloproteinase-2, 9 and vascular endothelial growth factor; and inhibited gastric cancer cell migration. In the xenograft mouse model, propranolol treatment significantly inhibited tumor growth, and immunohistochemistry revealed that propranolol led to the suppression of proliferation and induction of apoptosis. CONCLUSIONS: Propranolol inhibits the proliferation of gastric cancer cells by inducing G1-phase cell cycle arrest and apoptosis. These findings indicate that propranolol might have an opportunity as a new drug for gastric cancer.

The involvement of leucine-rich α-2 glycoprotein in the progression of skin and lung fibrosis in bleomycin-induced systemic sclerosis model.

OBJECTIVE: Systemc sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Recently, it has been shown that leucine-rich α-2 glycoprotein (LRG) functions as a modulator of transforming growth factor-ß (TGF-ß) signaling in fibrosis. We aimed to characterize the effect of LRG in SSc model and SSc patients. METHODS: Histological analysis was performed on LRG knockout (KO) and wild type (WT) mouse in the skin and the lung after bleomycin administration. Serum LRG levels were measured during the injection period. Gene expression analysis of the skin and lung tissue from LRG KO and WT mice was performed. In addition, serum LRG levels were determined in SSc patients and healthy controls. RESULTS: LRG KO mice display an inhibition of fibrosis in the skin in association with a decrease of dermal thickness, collagen deposition, and phospho-Smad3 expression after bleomycin. Serum LRG concentration significantly increased in WT mice after bleomycin. There was also a suppression of inflammation and fibrosis in the LRG KO mouse lung indicated by a reduction of lung weight, collagen content, and phospho-Smad3 expression after bleomycin. Gene expressions of TGF-ß and Smad2/3 were significantly reduced in LRG KO mice. Serum LRG levels in SSc patients were significantly higher than those in controls. CONCLUSION: LRG promotes fibrotic processes in SSc model through TGF-ß-Smad3 signaling, and LRG can be a biomarker for SSc in humans and also a potential therapeutic target for SSc.

TAS-116 inhibits oncogenic KIT signalling on the Golgi in both imatinib-naïve and imatinib-resistant gastrointestinal stromal tumours.

BACKGROUND: Despite the effectiveness of imatinib mesylate (IM), most gastrointestinal stromal tumours (GISTs) develop IM resistance, mainly due to the additional kinase-domain mutations accompanied by concomitant reactivation of KIT tyrosine kinase. Heat-shock protein 90 (HSP90) is one of the chaperone molecules required for appropriate folding of proteins such as KIT. METHODS: We used a novel HSP90 inhibitor, TAS-116, which showed specific binding to HSP90α/ß with low toxicity in animal models. The efficacy and mechanism of TAS-116 against IM-resistant GIST were evaluated by using IM-naïve and IM-resistant GIST cell lines. We also evaluated the effects of TAS-116 on the other HSP90 client protein, EGFR, by using lung cell lines. RESULTS: TAS-116 inhibited growth and induced apoptosis in both IM-naïve and IM-resistant GIST cell lines with KIT activation. We found KIT was activated mainly in intracellular compartments, such as trans-Golgi cisternae, and TAS-116 reduced autophosphorylated KIT in the Golgi apparatus. In IM-resistant GISTs in xenograft mouse models, TAS-116 caused tumour growth inhibition. We found that TAS-116 decreased phosphorylated EGFR levels and inhibited the growth of EGFR-mutated lung cancer cell lines. CONCLUSION: TAS-116 may be a novel promising drug to overcome tyrosine kinase inhibitor-resistance in both GIST and EGFR-mutated lung cancer.

Anti-glypican-1 antibody-drug conjugate is a potential therapy against pancreatic cancer.

BACKGROUND: Pancreatic cancer (PDAC) is the most lethal malignancy. New treatment options for it are urgently required. The aim was to develop an antibody-drug conjugate (ADC) targeting glypican-1 (GPC-1) as a new therapy for PDAC. METHODS: We evaluated GPC-1 expression in resected PDAC specimens and PDAC cell lines. We then measured the antitumour effect of anti-GPC-1 monoclonal antibody conjugated with the cytotoxic agent monomethyl auristatin F (MMAF) in vitro and in vivo. RESULTS: GPC-1 was overexpressed in most primary PDAC cells and tissues. The PDAC cell lines BxPC-3 and T3M-4 strongly expressed GPC-1 relative to SUIT-2 cells. Compared with control ADC, GPC-1-ADC showed a potent antitumour effect against BxPC-3 and T3M-4, but little activity against SUIT-2 cells. In the xenograft and patient-derived tumour models, GPC-1-ADC significantly and potently inhibited tumour growth in a dose-dependent manner. GPC-1-ADC-mediated G2/M-phase cell cycle arrest was detected in the tumour tissues of GPC-1-ADC-treated mice relative to those of control-ADC-treated mice. CONCLUSIONS: GPC-1-ADC showed significant tumour growth inhibition against GPC-1-positive pancreatic cell lines and patient-derived, GPC-1-positive pancreatic cancer tissues. Our preclinical data demonstrated that targeting GPC-1 with ADC is a promising therapy for patients with GPC-1-positive pancreatic cancer.

Epithelial-mesenchymal transition via transforming growth factor beta in pancreatic cancer is potentiated by the inflammatory glycoprotein leucine-rich alpha-2 glycoprotein.

We previously showed that an inflammation-related, molecule leucine-rich alpha-2 glycoprotein (LRG) enhances the transforming growth factor (TGF)-ß1-induced phosphorylation of Smad proteins and is elevated in patients with pancreatic ductal adenocarcinoma (PDAC). As TGF-ß/Smad signaling is considered to play a key role in epithelial-mesenchymal transition (EMT), we attempted to clarify the mechanism underlying LRG-related EMT in relation to metastasis in PDAC. We cultured LRG-overexpressing PDAC cells (Panc1/LRG) and evaluated the morphology, EMT-related molecules and TGF-ß/Smad signaling pathway in these cells. We also assessed the LRG levels in plasma and resected specimens from patients with PDAC. Inflammatory cytokines induced LRG production in PDAC cells. A spindle-like shape was visualized more frequently than other shapes in Panc1/LRG with TGF-ß1 exposure. The expression of E-cadherin in Panc1/LRG was decreased with TGF-ß1 exposure. Invasion increased with TGF-ß1 stimulation of Panc1/LRG. The phosphorylation of smad2 in Panc1/LRG was increased in comparison with parental Panc1 under TGF-ß1 stimulation. In the plasma LRG-high group, the recurrence rate tended to be higher and the recurrence-free survival (RFS) tended to be worse in comparison with the plasma LRG-low group. LRG enhanced EMT induced by TGF-ß signaling, thus indicating that LRG has a significant effect on the metastasis of PDAC.

Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer.

Glypican-1 (GPC1) is highly expressed in solid tumors, especially squamous cell carcinomas (SCCs), and is thought to be associated with disease progression. We explored the use of a GPC1-targeted antibody-drug conjugate (ADC) as a novel treatment for uterine cervical cancer. On immunohistochemical staining, high expression levels of GPC1 were detected in about 50% of uterine cervical cancer tissues and also in a tumor that had relapsed after chemoradiotherapy. Novel anti-GPC1 monoclonal antibodies were developed, and clone 01a033 was selected as the best antibody for targeted delivery of the cytotoxic agent monomethyl auristatin F (MMAF) into GPC1-positive cells. The anti-GPC1 antibody was conjugated with MMAF. On flow cytometry, HeLa and ME180 cervical cancer cells highly expressed GPC1, however, RMG-I ovarian clear cell cancer cell line showed weak expression. The GPC1-ADC was rapidly internalized into GPC1-expressing cells in vitro and was potently cytotoxic to cancer cells highly expressing GPC1. There were no inhibitory effects on cancer cells with low expression of GPC1. In a murine xenograft model, GPC1-ADC also had significant and potent tumor growth inhibition. GPC1-ADC-mediated G2/M phase cell cycle arrest was detected, indicating that the dominant antitumor effect in vivo was MMAF-mediated. The toxicity of GPC-ADC was tolerable within the therapeutic dose range in mice. Our data showed that GPC1-ADC has potential as a promising therapy for uterine cervical cancer.

Leucine rich α-2 glycoprotein is a potential urinary biomarker for renal tubular injury.

Recent evidence suggests that renal tubular injury plays a key role in deterioration of renal function in both chronic kidney disease (CKD) and acute kidney injury (AKI). Since commonly used biochemical indicators such as GFR, serum creatinine, blood urea nitrogen and creatinine clearance are inappropriate for detecting alteration in renal tubules, biomarkers reflecting renal tubular injury have been extensively explored. Our research group identified leucine rich α-2 glycoprotein (LRG) as a novel serum biomarker for various inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. In inflammatory diseases, LRG expression is up-regulated at the site of inflammation, in accordance with the induction of LRG in many cell types by various inflammatory cytokines. Recently, urinary LRG was reported as a possible biomarker for several renal diseases, but the mechanism of LRG excretion in urine is still unclear. In this study, by analyzing a mouse albumin (ALB) overload model that is commonly used to study proteinuria-induced renal tubular injury, we provided evidence that urinary LRG is produced in renal tubular epithelial cells by interleukin-1ß (IL-1ß) that is released during proteinuria-induced renal damage. In this model, urinary LRG became detectable after ALB overload. In kidney, mRNA expression of LRG together with that of NACHT LRR and PYD domains-containing protein 3 (NLRP3) and IL-1ß was significantly up-regulated in ALB-overloaded mice, compared to PBS-treated mice. By pathological analysis of kidney, LRG was detected in the injured proximal tubules, distal tubules and collecting ducts in ALB-overloaded mice. Accordingly, in vitro stimulation of mouse renal cortical tubular epithelial cells with excessive ALB led to LRG mRNA up-regulation and its protein secretion, which was effectively blocked by IL-1 receptor antagonist. These results suggest that urinary LRG could be applied to a biomarker detecting renal tubular injury in various renal diseases.

SOCS1 gene therapy has antitumor effects in imatinib-resistant gastrointestinal stromal tumor cells through FAK/PI3 K signaling.

BACKGROUND: Most of the gastrointestinal stromal tumors (GIST) have mutations in the KIT gene, encoding a receptor tyrosine kinase. Imatinib, a receptor tyrosine kinase inhibitor, is the first-line therapy for unresectable and metastatic GISTs. Despite the revolutionary effects of imatinib, some patients are primarily resistant to imatinib and many become resistant because of acquisition of secondary mutations in KIT. This study investigated the antitumor effects of SOCS1 gene therapy, which targets several signaling pathways. METHODS: We used GIST-T1 (imatinib-sensitive) and GIST-R8 (imatinib-resistant) cells. We infected both cell lines with an adenovirus expressing SOCS1 (AdSOCS1) and examined antitumor effect and mechanisms of its agent. RESULTS: The latter harboured with secondary KIT mutation and had imatinib resistance > 1000-fold higher than the former cells. We demonstrated that AdSOCS1 significantly decreased the proliferation and induced apoptosis in both cell lines. Moreover, SOCS1 overexpression inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3), AKT, and focal adhesion kinase (FAK) in both of them. Inhibition of JAK signaling did not affect the proliferation enough. However, inhibition of the FAK signaling with an FAK inhibitor or RNA interference significantly showed inhibitory effect on cell growth and suppressed the phosphorylation of AKT, indicating a cross-talk between the AKT and FAK pathways in both the imatinib-sensitive and imatinib-resistant GIST cells. CONCLUSIONS: Our results indicate that the activation of FAK signaling is critical for proliferation of both imatinib-sensitive and -resistant GIST cells and the interference with FAK/AKT pathway might be beneficial for therapeutic target.

Suppressor of cytokine signaling-1 gene therapy induces potent antitumor effect in patient-derived esophageal squamous cell carcinoma xenograft mice.

Chronic inflammation is involved in cancer growth in esophageal squamous cell carcinoma (ESCC), which is a highly refractory cancer with poor prognosis. This study investigated the antitumor effect and mechanisms of SOCS1 gene therapy for ESCC. Patients with ESCC showed epigenetics silencing of SOCS1 gene by methylation in the CpG islands. We infected 10 ESCC cells with an adenovirus-expressing SOCS1 (AdSOCS1) to examine the antitumor effect and mechanism of SOCS1 overexpression. SOCS1 overexpression markedly decreased the proliferation of all ESCC cell lines and induced apoptosis. Also, SOCS1 inhibited the proliferation of ESCC cells via multiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and focal adhesion kinase (FAK)/p44/42 mitogen-activated protein kinase (p44/42 MAPK). Additionally, we established two xenograft mouse models in which TE14 ESCC cells or ESCC patient-derived tissues (PDX) were subcutaneously implanted. Mice were intra-tumorally injected with AdSOCS1 or control adenovirus vector (AdLacZ). In mice, tumor volumes and tumor weights were significantly lower in mice treated with AdSOCS1 than that with AdLacZ as similar mechanism to the in vitro findings. The Ki-67 index of tumors treated with AdSOCS1 was significantly lower than that with AdLacZ, and SOCS1 gene therapy induced apoptosis. These findings demonstrated that overexpression of SOCS1 has a potent antitumor effect against ESCC both in vitro and in vivo including PDX mice. SOCS1 gene therapy may be a promising approach for the treatment of ESCC.

Overexpression of glypican-1 implicates poor prognosis and their chemoresistance in oesophageal squamous cell carcinoma.

BACKGROUND: Despite the recent improvements in multimodal therapies for oesophageal squamous cell carcinoma (ESCC), the prognosis remains poor. The identification of suitable biomarkers for predicting the prognosis and chemo-sensitivity is required to develop targeted treatments and improve treatment results. METHODS: Proteins highly expressed in ESCC cell lines compared with normal oesophageal cell lines were screened by isobaric tag for relative and absolute quantitation (iTRAQ). We identified glypican-1 (GPC1) as a novel molecule. The clinicopathological characteristics of GPC1 were evaluated by immunohistochemistry using ESCC specimens, and clinical parameters were assessed. The correlation between GPC1 expression levels and chemo-sensitivity were analysed in vitro. RESULTS: In the immunohistochemical assessment of 175 ESCC patients, 98.8% expressed GPC1. These patients demonstrated significantly poorer prognosis compared with patients with low-GPC1 expression by survival assay (P<0.001). Higher chemoresistance was observed in the GPC1 high-expression group. GPC1 expression levels positively correlated with chemo-sensitivity against cis-Diammineplatinum (II) dichloride (CDDP), and are potentially associated with anti-apoptotic function based on alterations in the MAPK downstream signalling pathway and Bcl-2 family member proteins. CONCLUSIONS: GPC1 is an independent prognostic factor in ESCC and is a critical molecule for altering the threshold of chemoresistance to CDDP.

Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas.

BACKGROUND: Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported. METHODS: We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis. RESULTS: We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01). CONCLUSIONS: We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.

Salt-inducible kinase 3 deficiency exacerbates lipopolysaccharide-induced endotoxin shock accompanied by increased levels of pro-inflammatory molecules in mice.

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll-like receptor 4 on macrophages, several signalling cascades co-operatively up-regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt-inducible kinase [SIK, a member of the AMP-activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up-regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over-expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin-6 (IL-6), nitric oxide (NO) and IL-12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up-regulated in SIK3-deficient thioglycollate-elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3-deficient TEPM suppressed the levels of the above-mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3-knockout (KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1-KO (n = 8), SIK2-KO (n = 9) and wild-type (n = 8 or 9) groups. In addition, SIK3-KO bone marrow transplantation increased LPS sensitivity of the recipient wild-type mice, which was accompanied by an increased level of circulating IL-6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS-stimulated macrophages.