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.

Nano-particulate Toll-like Receptor 9 Agonist Potentiates the Antitumor Activity of Anti-Glypican-1 Antibody.

BACKGROUND/AIM: Monoclonal antibodies (mAbs) that target tumor antigens have recently been developed. Their antitumor activity is mainly achieved through antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as tumor-infiltrated macrophages and natural killer (NK) cells. CpG oligodeoxynucleotides (ODNs) have potent antitumor activity and are considered to increase the tumor infiltration of macrophages and NK cells; however, a completely solubilized novel CpG-schizophyllan (SPG) complex, K3-SPG, displays more potent antitumor activity. We recently reported the significant antitumor activity of anti-glypican-1 (GPC1) mAb against GPC1-positive esophageal squamous cell carcinoma (ESCC) via ADCC. The aim of this study was to evaluate the potential synergistic antitumor activity of anti-GPC1 mAb and K3-SPG and elucidate the underlying mechanisms using a xenograft model of GPC1-positive human ESCC cells. MATERIALS AND METHODS: The established human esophageal cancer cell line TE14 was subcutaneously injected into SCID mice. Xenograft mice were treated with anti-GPC1 mAb, K3-SPG, or their combination. Antitumor activity was evaluated by measuring the tumor volume. For FACS analysis, agents were administrated, and tumors were resected 1 day after the final treatment. RESULTS: Anti-GPC1 mAb or K3-SPG monotherapy showed dose-dependent antitumor activity, and combination therapy with anti-GPC1 mAb and K3-SPG showed antitumor activity (p=0.0859). Flow cytometry revealed significantly increased numbers of macrophages (p=0.0133) and of the ratio of activated NK cells/total NK cells (p=0.0058) following K3-SPG or combination therapy. CONCLUSION: Combination therapy with K3-SPG and anti-GPC1 mAb or another antitumor mAb may represent a new cancer treatment option acting via ADCC.

Lipolysis-stimulated lipoprotein receptor-targeted antibody-drug conjugate demonstrates potent antitumor activity against epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is a lethal malignant tumor, for which new treatment options are urgently required. Lipolysis-stimulated lipoprotein receptor (LSR) is widely expressed in EOC, and it is associated with poor prognosis. In this study, we developed an antibody-drug conjugate (ADC) targeting LSR as a new therapeutic approach to EOC. METHODS: We, herein, developed novel anti-LSR monoclonal antibodies (mAbs) and an LSR-ADC by conjugating monomethyl auristatin E as a payload. We subsequently evaluated the in vitro and in vivo (on xenograft models) antitumor effect of the LSR-ADC. RESULTS: An overexpression of LSR was observed not only in the primary EOC tumor but also in its lymph node and omental metastases. The EOC cell lines NOVC7-C and OVCAR3 strongly expressed LSR (as compared to ES2 cells). Both the anti-LSR mAb and the LSR-ADC were able to specifically bind to LSR-positive cells and were rapidly internalized and trafficked to the lysosomes. The LSR-ADC demonstrated a potent antitumor effect against NOVC-7C and OVCAR3, but little activity against ES2 cells. In vitro, the LSR-ADC exhibited a potent antitumor effect against NOVC-7C and OVCAR3. Moreover, in the OVCAR3 xenograft models as well as in the patient-derived xenograft models of LSR-positive EOC, the LSR-ADC significantly inhibited tumor growth. The LSR-ADC also suppressed the omental/bowel metastases in OVCAR3-Luc xenografts and improved the median survival. CONCLUSION: The developed LSR-ADC demonstrated a significant antitumor activity against LSR-positive EOC cell lines and tumors. Our preclinical data support the use of the LSR-ADC as a novel therapy for patients with LSR-positive ovarian cancer.

SOCS1 Gene Therapy for Head and Neck Cancers: An Experimental Study.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is a fatal and debilitating disease, which is characterized by steady, poor survival rates despite advances in treatment. Suppressor of cytokine signaling (SOCS) 1 is up-regulated following cytokine-induced Janus kinase - signal transducer and activator of transcription (JAK-STAT) pathway activation, and inhibitors of cytokine signaling play roles in regulating cell growth and differentiation. We investigated the therapeutic potential of SOCS1 for HNSCC. MATERIALS AND METHODS: We used cell lines of oropharyngeal and tongue cancers (Detroit-562 and SCC-9, respectively) and a recombinant adenovirus vector expressing SOCS1 (AdSOCS1). RESULTS: AdSOCS1-induced SOCS1 overexpression significantly decreased cell proliferation through G2M phase cell cycle arrest and apoptosis. AdSOCS1 inhibited cell growth more strongly in SCC-9 cells than in Detroit-562 cells. JAK inhibitor I induced cell cycle arrest at the G0/G1 and GfM phases in Detroit-562 and SCC-9 cells, respectively. AdSOCS1 also decreased the activity of phosph-STAT3 (pSTAT3) and phosphop44/42 mitogen-activated protein kinase (p-p44/42 MAPK), as well as the expression of the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL). JAK inhibitor I decreased the expression of pSTAT3, but not p-p44/42 or Bcl-xL. The MAPK/extracellular signal-regulated kinase (MEK) inhibitor, U0126, decreased the expression of Bcl-xL in SCC-9 cells, but not in Detroit-562 cells. AdSOCS1 treatment inhibited tumor growth in mouse xenograft models. CONCLUSION: Overexpression of SOCS1 has a potent antitumor effect on HNSCC, suggesting the potential for clinical use. The varying effectiveness among cancer cells by over expression of SOCS1 may contribute to efficacy of SOCS 1 gene therapy for clinical use.

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.

Correction: Lipolysis-stimulated lipoprotein receptor overexpression is a novel predictor of poor clinical prognosis and a potential therapeutic target in gastric cancer.

[This corrects the article DOI: 10.18632/oncotarget.25952.].

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.

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.

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.

Lipolysis-stimulated lipoprotein receptor overexpression is a novel predictor of poor clinical prognosis and a potential therapeutic target in gastric cancer.

The prognosis of patients with advanced gastric cancer (GC) remains poor despite the recent advances in molecular targeted therapies, and the search for biomarkers that can predict prognosis and additional new agents with acceptable toxicity profiles are needed. Lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor that binds to triglyceride-rich lipoproteins and related to some malignancies. Herein, we examined the association between LSR expression and the prognosis of patients with GC, and investigated the antitumor effect of a previously developed anti-human LSR monoclonal antibody (#1-25). We first performed immunohistochemical analysis of LSR protein expression in GC and normal tissues, and then examined its association with the prognosis of 110 patients with GC. LSR was overexpressed in most of primary GC and metastatic tumors, but not in normal tissues. Patients with strong LSR expression (N = 80, 72.7%) had significantly poorer overall survival (OS) than those with weak expression (P = 0.017). Multivariate analysis identified strong LSR (as well as pT) as independent and significant prognostic factors for OS. Next, we demonstrated that very low density lipoprotein (VLDL) treatment increases cell proliferation in LSR-expressing GC cell lines in vitro; LSR inhibition using #1-25 inhibited VLDL-induced proliferation by suppressing JAK/STAT and PI3K signaling. In vivo, we demonstrated a marked antitumor effect of #1-25 in 2 distinct GC cell line xenograft mice models. Our findings suggest that LSR plays a key functional role in GC development, and that this antigen can be therapeutically targeted to improve GC treatment.

Intratumoral Delivery of an Adenoviral Vector Carrying the SOCS-1 Gene Enhances T-Cell-Mediated Antitumor Immunity By Suppressing PD-L1.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths and novel therapeutic strategies are required. Programmed cell death 1 and programmed cell death ligand 1 (PD-L1), which are key mediators of host immune tolerance, are associated with ovarian cancer progression. Recent evidence indicates the importance of IFNγ-induced PD-L1 for immune tolerance in ovarian cancer. This study aimed to reveal the therapeutic potential of suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of the Janus kinase (JAK)-STAT signaling pathway, for the treatment of ovarian cancer. IHC assessment revealed that patients with ovarian cancer with high intratumoral STAT1 activation exhibited poor prognosis compared with patients with low STAT1 activation (P < 0.05). Stimulation of OVISE, OVTOKO, OV2944-HM-1 (HM-1), and CT26 cell lines with IFNγ induced STAT1 phosphorylation and PD-L1 expression. Adenovirus-mediated SOCS-1 gene delivery (AdSOCS-1) in HM-1 and CT26 cells in vitro potently inhibited IFNγ-induced STAT1 phosphorylation and PD-L1 upregulation, similar to the addition of JAK inhibitor I, but failed to inhibit their proliferation. Notably, intratumoral injection of AdSOCS-1, but not AdLacZ, significantly inhibited the tumor growth of HM-1 and CT26 cells subcutaneously transplanted in immunocompetent syngeneic mice. AdSOCS-1 reduced PD-L1 expression on tumors and restored the activation of tumor-infiltrating CD8+ T cells. Moreover, the antitumor effect of AdSOCS-1 was significantly attenuated by PD-L1 Fc-fusion protein administration in vivo, suggesting that the effect of AdSOCS-1 is mainly attributable to enhancement of tumor immunity. This study highlights the potential clinical utility of SOCS-1 as an immune checkpoint inhibitor. Mol Cancer Ther; 17(9); 1941-50. ©2018 AACR.

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.

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.

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 promotes lung fibrosis by modulating TGF-ß signaling in fibroblasts.

TGF-ß has an important role in fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Detailed analysis of TGF-ß signaling in pulmonary fibrosis at the molecular level is needed to identify novel therapeutic targets. Recently, leucine-rich alpha-2 glycoprotein (LRG) was reported to function as a modulator of TGF-ß signaling in angiogenesis and tumor progression. However, the involvement of LRG in fibrotic disorders, including IPF, has not yet been investigated. In this study, we investigated the role of LRG in fibrosis by analyzing LRG knockout (KO) mice with bleomycin-induced lung fibrosis, an animal model of pulmonary fibrosis. The amount of LRG in the lungs of wild-type (WT) mice was increased by bleomycin administration prior to fibrosis development. In LRG KO mice, lung fibrosis was significantly suppressed, as indicated by attenuated Masson's trichrome staining and lower collagen content than those in WT mice. Moreover, in the lungs of LRG KO mice, phosphorylation of Smad2 was reduced and expression of α-SMA was decreased relative to those in WT mice. In vitro experiments indicated that LRG enhanced the TGF-ß-induced phosphorylation of Smad2 and the expression of Serpine1 and Acta2, the downstream of Smad2, in fibroblasts. Although endoglin, an accessory TGF-ß receptor, is essential for LRG to promote TGF-ß signaling in endothelial cells during angiogenesis, we found that endoglin did not contribute to the ability of LRG to enhance Smad2 phosphorylation in fibroblasts. Taken together, our data suggest that LRG promotes lung fibrosis by modulating TGF-ß-induced Smad2 phosphorylation and activating profibrotic responses in fibroblasts.

SOCS1 Gene Therapy Improves Radiosensitivity and Enhances Irradiation-Induced DNA Damage in Esophageal Squamous Cell Carcinoma.

STAT3 has been implicated recently in radioresistance in cancer. In this study, we investigated the association between STAT3 and radioresistance in esophageal squamous cell carcinoma (ESCC). Strong expression of activated phospho-STAT3 (p-STAT3) was observed in 16/22 ESCC patients with preoperative chemoradiotherapy (CRT), compared with 9 of 24 patients with surgery alone, where the prognosis of those with CRT was poor. Expression of p-STAT3 and the antiapoptotic proteins Mcl-1 and survivin was strongly induced in ESCC cells by irradiation. Ectopic STAT3 expression increased radioresistance, whereas expression of the STAT3 negative regulator SOCS1 via an adenoviral vector improved radioresponse. Inhibiting the STAT3-Mcl-1 axis by SOCS1 enhanced DNA damage after irradition and induced apoptosis. Combining SOCS1 with radiotherapy enhanced antitumor responses in a murine xenograft model compared with the individual therapies. Tumor repopulation occurred transiently after treatment by irradiation but not the combination SOCS1/radiotherapy. Tumors subjected to this combination expressed high levels of γH2AX and low levels of Ki-67, which was maintained after cessation of treatment. Overall, we demonstrated that inhibiting the STAT3-Mcl-1 signaling axis by ectopic SOCS1 improved radiosensitivity by inducing apoptosis and enhancing DNA damage after radiotherapy, offering a mechanistic rationale for a new ESCC treatment. Cancer Res; 77(24); 6975-86. ©2017 AACR.

Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-ß-Smad2 signaling in naïve helper T cells.

BACKGROUND: Leucine-rich alpha 2 glycoprotein (LRG) has been identified as a serum protein elevated in patients with active rheumatoid arthritis (RA). Although the function of LRG is ill-defined, LRG binds with transforming growth factor (TGF)-ß and enhances Smad2 phosphorylation. Considering that the imbalance between T helper 17 (Th17) cells and regulatory T cells (Treg) plays important roles in the pathogenesis of RA, LRG may affect arthritic pathology by enhancing the TGF-ß-Smad2 pathway that is pivotal for both Treg and Th17 differentiation. The purpose of this study was to explore the contribution of LRG to the pathogenesis of arthritis, with a focus on the role of LRG in T cell differentiation. METHODS: The differentiation of CD4 T cells and the development of collagen-induced arthritis (CIA) were examined in wild-type mice and LRG knockout (KO) mice. To examine the influence of LRG on T cell differentiation, naïve CD4 T cells were isolated from LRG KO mice and cultured under Treg- or Th17-polarization condition in the absence or presence of recombinant LRG. RESULTS: In the CIA model, LRG deficiency led to ameliorated arthritis and reduced Th17 differentiation with no influence on Treg differentiation. By addition of recombinant LRG, the expression of IL-6 receptor (IL-6R) was enhanced through TGF-ß-Smad2 signaling. In LRG KO mice, the IL-6R expression and IL-6-STAT3 signaling was attenuated in naïve CD4 T cells, compared to wild-type mice. CONCLUSIONS: Our findings suggest that LRG upregulates IL-6R expression in naïve CD4 T cells by the enhancement of TGF-ß-smad2 pathway and promote Th17 differentiation and arthritis development.

Glypican-1 targeted antibody-based therapy induces preclinical antitumor activity against esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis despite the development of multimodal therapy. Expression of glypican-1 (GPC1) has been reported to be elevated in a subset of patients with ESCC and associated with chemoresistance. This study aimed to determine the association of GPC1 with ESCC growth and potential usefulness of the GPC1 targeted therapy by monoclonal antibody (mAb) in ESCC. Expression of GPC1 was higher in ESCC tumor tissues than in adjacent non-tumoral tissues and normal tissues. Knockdown of GPC1 decreased growth of ESCC cells and induced apoptosis via inhibition of EGFR, AKT and p44/42-MAPK signaling pathways in vitro. Anti-GPC1 mAb strongly inhibited tumor growth via antibody-dependent cellular cytotoxicity dependent and independent manner in GPC1-positive ESCC xenograft models. Anti-GPC1 mAb also inhibited tumor growth of GPC1 positive ESCC patients derived tumor xenograft models. Furthermore, anti-GPC1 mAb showed a significant tumor growth inhibition with decreased angiogenesis compared with IgG treated controls in ESCC xenografted mice. Treatment with anti-GPC1 mAb was not toxic in mice. Anti-GPC1 mAb may have a potent anti-tumor effect and represent a novel treatment option for patients with GPC1-positive ESCC.

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.