Antagonist of nucleolin, N6L, inhibits neovascularization in mouse models of retinopathies.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas.

Genetic alterations define different molecular subclasses of hepatocellular adenoma (HCA) linked with risk factors, histology and clinical behavior. Recently, Argininosuccinate Synthase 1 (ASS1), a major periportal protein, was proposed as a marker of HCA with a high risk of hemorrhage. We aimed to assess the significance of ASS1 expression through the scope of the HCA molecular classification. ASS1 expression was evaluated using RNAseq, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Immunohistochemistry. ASS1 and glioma-associated oncogene 1 (GLI1) expression were analyzed in vitro after modulation of GLI1 expression. Using RNAseq in 27 HCA and five nontumor liver samples, ASS1 expression was highly correlated with GLI1 expression (P<0.0001, R=0.75). In the overall series of 408 HCA, ASS1 overexpression was significantly associated with sonic hedgehog HCA (shHCA) compared to other molecular subgroups (P<0.0001), suggesting that sonic hedgehog signaling controls ASS1 expression. GLI1 expression silencing by siRNA induced a downregulation of ASS1 in PLC/PFR5 and SNU878 cell lines. In 390 HCA, we showed that ASS1 expression belonged to the periportal expression program that was maintained in shHCA but down-regulated in all the other HCA subtypes. In contrast, HCA with ß-catenin activation showed an activation of a perivenous program. Despite the significant association between GLI1 and ASS1 expression, ASS1 mRNA expression was not associated with specific clinical features. At the protein level using immunohistochemistry, prostaglandin D synthase (PTGDS) was strongly and specifically overexpressed in shHCA. CONCLUSION: ASS1 is associated with sonic hedgehog activation as part of a periportal program expressed in shHCA, a molecular subgroup defined by INHBE-GLI1 gene fusion. (Hepatology 2018).

Molecular Subtypes of Clear-cell Renal Cell Carcinoma are Prognostic for Outcome After Complete Metastasectomy.

BACKGROUND: Metastasectomy is routinely performed in selected patients with metastatic clear-cell renal cell carcinoma (ccRCC) as an alternative to systemic therapy. In the absence of randomized trials, the benefit and best way of patient selection remain unclear. Earlier, we described four molecular ccRCC-subtypes (ccrcc1-4) that have a prognostic and predictive value upon first-line sunitinib or pazopanib. OBJECTIVE: Assess the prognostic value of ccrcc1-4 subtypes after complete metastasectomy. (1) Compare outcomes of good-prognosis ccrccc2&3-tumors with intermediate/poor-prognosis ccrcc1&4-tumors. (2) Compare outcomes of the four subtypes separately. DESIGN, SETTING, AND PARTICIPANTS: Single-center retrospective study (1995-2017), assessing 43 ccRCC patients undergoing complete metastasectomy without systemic treatment. INTERVENTION: Molecular subtype determined with established 35-gene expression classifier. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Median disease-free survival (DFS), time to systemic therapy, cancer-specific (CSS) and overall survival (OS) from metastasectomy, estimated with Kaplan-Meier method and tested against other predictors with multivariable Cox regression. RESULTS AND LIMITATIONS: Median DFS was 23 mo for ccrcc2&3-tumors versus 9 mo for ccrcc1&4-tumors (p=0.011, hazard ratio [HR]=2.6). Median time to systemic therapy was 92 mo versus 28 mo (p=0.003, HR=3.3). Median CSS was 133 mo versus 50 mo (p<0.001, HR=2.7). Median OS was 127 mo versus 50 mo (p=0.011, HR=2.5). The classification remained independent upon multivariable analysis. Outcomes remained significantly different when comparing four subtypes separately. The intrinsic heterogeneity of expression profiles is a limitation of this approach. CONCLUSION: Even after clinical patient selection, patients with a ccrcc1- or ccrcc4-tumor are at a higher risk of relapse after complete metastasectomy. Patients with a ccrcc2- or ccrcc3-tumor usually experience a long DFS. These results need validation in a larger cohort to establish the subtypes as prognostic marker. PATIENT SUMMARY: Metastasectomy is recommended for some patients with metastatic clear-cell kidney cancer; however, we do not know who will benefit the most. We show that molecular subtypes increase the possibility to predict which patients are at risk for early relapse after metastasectomy and who may benefit more from other treatment options.

Molecular Subtypes of Clear Cell Renal Cell Carcinoma Are Associated With Outcome During Pazopanib Therapy in the Metastatic Setting.

BACKGROUND: We previously described 4 molecular subtypes of metastatic clear cell renal cell carcinoma (mccRCC), named ccrcc1-4 (Beuselinck et al, 2015). These have both prognostic and predictive value for patients treated with first-line sunitinib, with distinctive objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). The ccrcc2 and ccrcc3 tumors have the best outcomes, followed by ccrcc1 and then ccrcc4. We hypothesized that these molecular subtypes would show similar outcomes with first-line pazopanib treatment. PATIENTS AND METHODS: We classified 28 mccRCC tumors treated with pazopanib as first-line therapy, as described previously. The primary endpoints were PFS and OS from the start of pazopanib. A secondary endpoint was ORR. Because there were only 2 ccrcc3 tumors, they were pooled with the ccrcc2 tumors for outcome analysis. RESULTS: PFS was 9 months for the ccrcc2 and ccrcc3 tumors, 5 months for ccrcc1 tumors, and 3 months for the ccrcc4 tumors (P = .011). The corresponding OS duration was 69, 19, and 5 months (P = .003). The corresponding ORR was 50%, 33%, and 0%. The corresponding mean tumor size decreased by 34%, 6%, and 2% (P = .032). The ccrcc1-4 classification was a stronger predictor of outcome than the International Metastatic Renal Cell Carcinoma Database Consortium score on univariate analysis (P = .011 vs. P = .094 for PFS and P = .003 vs. .013 for OS). Both remained independent on bivariate analysis. CONCLUSION: The molecular subtypes of mccRCC are associated with outcome on pazopanib as first-line therapy. The prognostic and predictive value of the ccrcc1-4 molecular classification requires validation in prospective trials.

Nucleolin Targeting Impairs the Progression of Pancreatic Cancer and Promotes the Normalization of Tumor Vasculature.

Pancreatic cancer is a highly aggressive tumor, mostly resistant to the standard treatments. Nucleolin is overexpressed in cancers and its inhibition impairs tumor growth. Herein, we showed that nucleolin was overexpressed in human specimens of pancreatic ductal adenocarcinoma (PDAC) and that the overall survival significantly increased in patients with low levels of nucleolin. The nucleolin antagonist N6L strongly impaired the growth of primary tumors and liver metastasis in an orthotopic mouse model of PDAC (mPDAC). Similar antitumor effect of N6L has been observed in a highly angiogenic mouse model of pancreatic neuroendocrine tumor RIP-Tag2. N6L significantly inhibited both human and mouse pancreatic cell proliferation and invasion. Notably, the analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion both in mPDAC and RIP-Tag2 tumors, in parallel to an inhibition of tumor hypoxia. Nucleolin inhibition directly affected endothelial cell (EC) activation and changed a proangiogenic signature. Among the vascular activators, nucleolin inhibition significantly decreased angiopoietin-2 (Ang-2) secretion and expression in ECs, in the tumor and in the plasma of mPDAC mice. As a consequence of the observed N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the antitumor properties of gemcitabine in PDAC mice. In conclusion, nucleolin inhibition is a new anti-pancreatic cancer therapeutic strategy that dually blocks tumor progression and normalizes tumor vasculature, improving the delivery and efficacy of chemotherapeutic drugs. Moreover, we unveiled Ang-2 as a potential target and suitable response biomarker for N6L treatment in pancreatic cancer. Cancer Res; 76(24); 7181-93. ©2016 AACR.