FGF2 alters macrophage polarization, tumour immunity and growth and can be targeted during radiotherapy.

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2LMW-/- mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS+/CD206+ TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.

Type I IFN protects cancer cells from CD8+ T cell-mediated cytotoxicity after radiation.

Treatment of tumors with ionizing radiation stimulates an antitumor immune response partly dependent on induction of IFNs. These IFNs directly enhance dendritic cell and CD8+ T cell activity. Here we show that resistance to an effective antitumor immune response is also a result of IFN signaling in a different cellular compartment of the tumor, the cancer cells themselves. We abolished type I IFN signaling in cancer cells by genetic elimination of its receptor, IFNAR1. Pronounced immune responses were provoked after ionizing radiation of tumors from 4 mouse cancer cell lines with Ifnar1 knockout. This enhanced response depended on CD8+ T cells and was mediated by enhanced susceptibility to T cell-mediated killing. Induction of Serpinb9 proved to be the mechanism underlying control of susceptibility to T cell killing after radiation. Ifnar1-deficient tumors had an augmented response to anti-PD-L1 immunotherapy with or without radiation. We conclude that type I IFN can protect cancer cells from T cell-mediated cytotoxicity through regulation of Serpinb9. This result helps explain why radiation of tumors can stimulate antitumor immunity yet also result in resistance. It further suggests potential targets for intervention to improve therapy and to predict responses.

STING-Dependent Interferon-λ1 Induction in HT29 Cells, a Human Colorectal Cancer Cell Line, After Gamma-Radiation.

PURPOSE: To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays. METHODS AND MATERIALS: Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown. RESULTS: Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis. CONCLUSIONS: Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment.

Expression levels of serum miRNA-195 in different types of patients with cholangiocarcinoma and its value to determine the prognosis thereof.

This study aimed to investigate the expression levels of microRNA-195 (miRNA-195) in different types of patients with cholangiocarcinoma (CCA) and its correlation with the prognosis. Serum samples were collected from different types of patients with CCA (I, II, III, IV) and normal cases, followed by detection of expression of miRNA-195 using quantitative polymerase chain reaction (qPCR). The serum samples of 204 patients with CCA, including 75 cases of type I, 68 cases of type II, 35 cases of type III and 26 cases of type IV and 200 healthy subjects were selected. The baseline clinicopathological data of patients with CCA were assessed and recorded, and patients were followed up constantly. The receiver operating characteristic (ROC) curve was established, and the area under the ROC curve (AUC) was calculated to evaluate the difference of miRNA-195 expression levels between patients with CCA and normal controls. Survival curves were set up for groups with high and low expression levels via the Kaplan-Meier method, and the log-rank test was used to evaluate the difference of survival curves between the two groups. The expression of miR-195 in patients with CCA was significantly lower than that in the normal control group, with a sensitivity of 0.78 and a specificity of 0.76, and it was positively correlated with the pathological grade of CCA. Additionally, the expression level of serum miRNA-195 was associated with lymph node metastasis (P=0.009) and tumor-node-metastasis (TNM) classification (P=0.010). The survival analysis revealed that the prognosis in patients with CCA in types III and IV was poorer than that in those with types I and III who had a low expression of miRNA-195 (P=0.0026). The results show that miR-195 is an important marker that reflects the malignant degree of CCA, and it is expected to be a reference marker to determine the prognosis of CCA.

Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2-dependent angiogenesis in mice.

Hepatic metastases are amenable to ablation; however, many patients are not suitable candidates for such therapy and recurrence is common. The tumor microenvironment is known to be essential for metastatic growth, yet identification of plausible targets for cancer therapy in the microenvironment has proven elusive. We found that human colorectal cancer liver metastases and murine gastrointestinal experimental liver metastases are infiltrated by neutrophils. Plasticity in neutrophils has recently been shown to lead to both protumor and antitumor effects. Here, neutrophils promoted the growth of hepatic metastases, given that depletion of neutrophils in already established, experimental, murine liver metastases led to diminished metastatic growth. Decreased growth was associated with reductions in vascular density and branching suggestive of vessel normalization. Metastasis-associated neutrophils expressed substantially more fibroblast growth factor 2 (FGF2) than naïve neutrophils, indicating neutrophil polarization by the tumor microenvironment. Administration of FGF2 neutralizing antibody to mice bearing experimental liver metastases phenocopied neutrophil depletion by reducing liver metastatic colony growth, vascular density, and branching. CONCLUSION: Here, we show, using FGF2 as an example, that identification of factors responsible for the protumoral effects of infiltrating myeloid cells can be used to target established liver metastases. Such therapies could be utilized to limit disease progression and potentiate the effects of standard ablative therapies. (Hepatology 2017;65:1920-1935).

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Recruitment of myeloid cells to the tumor microenvironment supports liver metastasis.

Tumor-infiltrating immune cells play important roles in metastasis. We have recently revealed the recruitment of a specific myeloid cell subset (CD11b/Gr1mid) to hepatic metastases. Such a recruitment relies on CCL2/CCR2 signaling and acts to sustain metastatic growth. A similar cell subset was identified in patients bearing hepatic metastases of colorectal cancer, highlighting the potential therapeutic relevance of our findings.

Recruitment of a myeloid cell subset (CD11b/Gr1 mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis.

UNLABELLED: Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1(mid) cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor-derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1(mid) recruitment and decreased tumor burden. Depletion of the CD11b/Gr1(mid) subset in a transgenic CD11b-diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1(mid) cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. CONCLUSION: Collectively, our findings highlight the importance of myeloid cells--in this case a selective CD11b/Gr1(mid) subset--in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy.

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

[Association between anti-endothelial cell antibody and response to dexamethasone in sudden hearing loss].

OBJECTIVE: To investigate relationship between anti-endothelial cell antibody(AECA) and response to dexamethasone in sudden hearing loss(SHL). METHOD: Forty-eight SHL patients and thirty normal controls with SHL were recruited in present study. AECA was detected by ELISA in serum of all normal controls and SHL patients as well as pure-tone average was examined by electronic audiometry during treatment in SHL patients. Both AECA-positive and -negative subjects received 10 mg/d venous dexamethasone for 5 days followed by gradual tapering of dose of 5 mg/d for another 5-day. Then pure-tone average was reexamined. Differences in hearing recovery between AECA-positive and -negative subjects and relationship between AECA level and hearing recovery were analyzed. RESULT: The prevalence of AECA detection was 68.75% (33 of 48 patients) in SHL patients, with significant difference compared with control subjects with 23.33% (7 of 30 controls) (P<0.01). After treatment, rates of response to dexamethasone in AECA-positive and -negative SHL patients were 81.8% (27 of 33 patients) and 33.3% (5 of 15 patients), respectively. Meanwhile, there was a significant difference in cure, excellent recovery, partly recovery and invalid between AECA-positive and -negative groups [21.2% (7/33), 33.3% (11/33), 27.3% (9/33) and 18.2% (6/33) versus 0, 13.3% (2/15), 20.0% (3/15) and 66.7% (10/15), P<0.01]. Except 5 subjects with AECA level more than 263 microg/L, hearing recovery was correlated to pretreatment AECA level (r=0.8084, P<0.01). CONCLUSION: In sudden HL patients treated with dexamethasone, AECA might represent a serological marker of prognosis.

The centromere site of the segregation cassette of broad-host-range plasmid RA3 is located at the border of the maintenance and conjugative transfer modules.

RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative -35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ∼300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ∼300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.

Matrix metalloproteinase-9 regulates tumor cell invasion through cleavage of protease nexin-1.

Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1.

Hyperactivation of mammalian target of rapamycin (mTOR) signaling by a gain-of-function mutant of the Rheb GTPase.

Gain-of-function mutants of Ras and Rho family small GTPases have proven to be important tools in analyzing signaling downstream of these small GTPases. The Ras-related GTPase Rheb has emerged as a key player downstream of TSC1-2 in activating signaling to mammalian target of rapamycin (mTOR) effectors of cell growth such as S6K and 4E-BP1. The TSC1-2 tumor suppressor complex has been shown to act as a RhebGAP, converting Rheb from a GTP-bound to a GDP-bound form. Here we report the identification of a mutant Rheb (S16HRheb) that exhibits gain-of-function properties. At endogenous levels of expression S16HRheb exhibits increased GTP loading in vivo and is resistant to TSC1-2 GAP in vitro. Compared with wild-type Rheb, S16HRheb is more active at promoting the phosphorylation of the mTOR effectors S6K1 and 4E-BP1. Thus S16HRheb will help to identify proximal signaling events downstream of Rheb and allow potential Rheb-independent functions downstream of TSC1-2 to be investigated.