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Additional biomarkers for the development and progression of colorectal cancer (CRC) remain to be identified. Hence, the current study aimed to identify potential diagnostic markers for CRC. Analyses of cysteine protease inhibitor [cystatins (CSTs)] expression in CRC samples and its correlation with cancer stage or survival in patients with CRC demonstrated that CRC tissues had greater CST1 and CST2 mRNA expression compared to noncancerous adjacent tissues, while higher CST2 mRNA expression in CRC tissues was correlated with advanced stages and disease-free survival in patients with CRC, encouraging further exploration on the role of CST2 in CRC. Through an online database search and tissue microarray (TMA), we confirmed that CRC samples had higher CST2 expression compared to noncancerous adjacent tissue or normal colorectal tissues at both the mRNA and protein levels. TMA also revealed that colorectal adenoma, CRC, and metastatic CRC tissues exhibited a significantly increased CST2 protein expression. Accordingly, survival analysis demonstrated that the increase in CST2 protein expression was correlated with shorter overall survival of patients with CRC. Moreover, our results found a significant upregulation of CST2 in multiple cancer tissues. Taken together, these findings suggest the potential role of CST2 as a diagnostic and prognostic biomarker for CRC.

Background: NUFIP1 is an RNA-binding protein that interacts with fragile X mental retardation protein (FMRP) in the messenger ribonucleoprotein particle (mRNP). We previously showed that NUFIP1 was upregulated in colorectal cancer (CRC), but how the protein may contribute to the disease and patient prognosis is unknown. Methods: Here we combine database analysis, microarray, quantitative PCR, and immunohistochemistry of patients' samples to confirm our previous findings on NUFIP1 overexpression in CRC, and to reveal that increased expression of NUFIP1 in CRC tissues correlated with worse overall, recurrence-free, event-free and disease-free survival in patients, as well as with more advanced CRC clinicopathological stage. Results: Loss of function analysis demonstrated that NUFIP1 knockdown suppressed cell growth in vitro and in vivo, inhibited cell viability and survival, and induced cell cycle arrest and apoptosis in vitro. In addition, as a natural anticancer triterpene from various fruits and vegetables, ursolic acid (UA) treatment suppressed cell proliferation, down-regulated NUFIP1 protein expression, and further enhanced the effects of NUFIP1 knockdown in CRC cells in vitro. NUFIP1 knockdown up-regulated the expression of 136 proteins, down-regulated the expression of 41 proteins, and enriched multiple signaling pathways including the senescence-associated heterochromatin foci (SAHF) pathway. Furthermore, NUFIP1 knockdown enhanced the expression of senescence-associated-β-galactosidase (SA-β-gal), the SAHF markers HP1γ and trimethylation (H3k9me3), and the senescence-related protein HMGA2. Conclusion: Our findings suggest that NUFIP1 is overexpressed in CRC and correlates with disease progression and poor patient survival. NUFIP1 may exert oncogenic effects partly by altering senescence. UA may show potential to treat CRC by down-regulating NUFIP1.

Blood vessels in the tumor periphery have high pericyte coverage and are resistant to vascular disrupting agents (VDAs). VDA treatment resistance leads to a viable peripheral tumor rim that contributes to treatment failure and disease recurrence. Here, we provide evidence to support a hypothesis that shifting the target of VDAs from tumor vessel endothelial cells to pericytes disrupts tumor peripheral vessels and the viable rim, circumventing VDA treatment resistance. Through chemical engineering, we developed Z-GP-DAVLBH (from the tubulin-binding VDA desacetylvinblastine monohydrazide [DAVLBH]) as a prodrug that can be selectively activated by fibroblast activation protein α (FAPα) in tumor pericytes. Z-GP-DAVLBH selectively destroys the cytoskeleton of FAPα-expressing tumor pericytes, disrupting blood vessels both within the core and around the periphery of tumors. As a result, Z-GP-DAVLBH treatment eradicated the otherwise VDA-resistant tumor rim and led to complete regression of tumors in multiple lines of xenografts without producing the drug-related toxicity that is associated with similar doses of DAVLBH. This study demonstrates that targeting tumor pericytes with an FAPα-activated VDA prodrug represents a potential vascular disruption strategy in overcoming tumor resistance to VDA treatments.

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters is the major obstacle for chemotherapeutic success. Although attempts have been made to circumvent ABC transporter-mediated MDR in past decades, there is still no effective agent in clinic. Here, we identified a vascular disrupting agent, Z-GP-DAVLBH, that significantly inhibited the growth of multidrug-resistant human hepatoma HepG2/ADM and human breast cancer MCF-7/ADR tumor xenografts, although these cells were insensitive to Z-GP-DAVLBH in vitro. Z-GP-DAVLBH increased the secretion of granulocyte-macrophage colony-stimulating factor in tumor tissues and serum of tumor-bearing mice to skew tumor-associated macrophages from the pro-tumor M2 phenotype to the antitumor M1 phenotype, thereby contributing to the induction of HepG2/ADM and MCF-7/ADR cell apoptosis. Our findings shed new light on the underlying mechanisms of VDAs in the treatment of drug-resistant tumors and provide strong evidence that Z-GP-DAVLBH should be a promising agent for overcoming MDR.

Background and Objective: Tumor angiogenesis process is regulated by multiple proangiogenic pathways, such as VEGFR2 and Axl. Inhibition of VEGF/VEGFR2 signaling alone fails to block tumor neovascularization, and anti-VEGF resistance is often associated with Axl. Hence, discovery of novel agents that target multiple angiogenesis pathways is in demand. Here, we describe desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB) that exerts a more potent antiangiogenic effect than VLB in vitro and in vivo by inhibiting VEGFR2 and Axl pathways. Methods: The antiangiogenic effects of DAVLBH were studied in vitro (proliferation, migration, and tube formation assays) and ex vivo (aortic ring assay). In vitro pericyte migration to endothelial tubes was assessed using a three-dimensional co-culture assay. In vivo assay was performed in HeLa xenograft model. Western blotting, immunohistochemical and immunofluorescence assays were conducted to evaluate the key proteins of the VEGF/VEGFR2 and Gas6/Axl pathways. Results: DAVLBH (1 nM) inhibited VEGF- and Gas6-induced angiogenesis in vitro. At 0.75 μmol/kg, DAVLBH significantly delayed tumor growth and reduced vascular density in vivo, which was associated with the inactivation of VEGF/VEGFR2 and Gas6/Axl signalling pathways. DAVLBH blocked the compensatory upregulation of Axl in response to bevacizumab treatment in HUVECs. DAVLBH also suppressed the recruitment of pericytes to well-established endothelial tubes and reduced pericyte coverage in vivo, which was accompanied by inhibition of Axl. Conclusions: DAVLBH potently inhibited angiogenesis-mediated tumor growth by blocking the activation of VEGF/VEGFR2 and Gas6/Axl pathways. DAVLBH might serve as a promising antiangiogenic agent for cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Minfeng Chen, Xueping Lei, Qiulin Nie, Jianyang Hu, Zhenjian Zhuo, Anita Yiu, Heru Chen, Nanhui Xu, Maohua Huang, Kaihe Ye, Liangliang Bai, Wencai Ye, Dong-Mei Zhang. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2017-1812