Daniel J. Fernandes

The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells.

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.

Plasma Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in MV4-11 Leukemia Cells

AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [32P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [32P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 ± 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of [32P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.

Mechanism of Regulation of bcl-2 mRNA by Nucleolin and A+U-rich Element-binding Factor 1 (AUF1)

The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3′-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3′-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5′ end of the 136-nucleotide bcl-2 AU-rich element (AREbcl-2). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to AREbcl-2. In RNA decay assays, AREbcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of AREbcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

Regulation of Bcl-2 Expression by HuR in HL60 Leukemia Cells and A431 Carcinoma Cells

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3′-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability. (Mol Cancer Res 2009;7(8):1354–66)

Understanding the actions of carcinostatic drugs to improve chemotherapy: 5-fluorouracil

Abstract The accumulation of additional basic knowledge about 5-fluorouracil is still required in order to improve the optimal utilization of this drug in the cancer patient. The drugs inhibitory effects on ribosome formation, already recognized as an important drug response in microorganisms and rodent tissues, has now been demonstrated for human colon tumors. However, ribosome synthesis does not occur sufficiently rapidly under the conditions of these experiments so that this assay appears to have limited promise as a predictor for human tumor responsiveness to FU. In the L1210 system, a rodent tumor model sensitive to FU, FdUMP was formed rapidly, and this metabolite persisted for several days in the tumor after a single dose of FU. DNA synthesis was inhibited simultaneously in concurrence with peak FdUMP levels, and recovery of this inhibition took place as the tissue concentration of FdUMP declined. DNA synthesis in bone marrow and gastrointestinal tract, which was also inhibited by drug treatment, recovered more rapidly than that of tumor, in line with the antitumor selectivity of FU in this tumor model. For the relatively unresponsive Walker 256 rat tumor, even higher levels of FdUMP were formed initially, but this metabolite was retained only briefly in the tumor after a single dose of FU. Inhibition of DNA formation in tumor was more transient than that in normal tissues, probably accounting for the nonselective antitumor response in that model. It is likely that tissue retention of FdUMP may serve as a major determinant for activity of FU in tumor and normal tissues, but other factors including inhibition of ribosomal maturation also play a role in the drugs cytotoxic action.

Abstract 1488: Development of anti-nucleolin antibodies with broad spectrum anticancer activity and negligible toxicity to normal cells

Nucleolin has multiple, unique functions in cancer cells, including the shuttling of ligands from the cell surface to the cytoplasm and the stabilization of oncogene and cytokine mRNAs that have an AU-rich nucleolin binding element in their 3’-UTRs. We developed a panel of anti-nucleolin antibodies that exploit the temperature-dependent shuttling function of nucleolin to gain access to the cytoplasm of human tumor cells and induce oncogene mRNA destabilization. Several lines of evidence indicate that our fully human monoclonal IgG1 antibody, CP101.2C8, targets nucleolin and penetrates tumor cells. CP101.2C8 bound tightly to human recombinant nucleolin (Kd = 26 ± 7 nM, SEM.) and to plasma membrane nucleolin of human tumor cells. Confocal microscopy of Panc-1 and DU-145 tumor cells incubated at 37 0C with CP101.2C8 revealed punctate localization of the antibody in the plasma membranes of these cells and internalization of the antibody into the cytoplasm. The localization of the antibody within foci in the plasma membrane suggested that the antibody was bound to nucleolin that was incorporated into lipid rafts within the plasma membrane. MCF-7 human breast cancer cells were made more than 100-fold resistant to CP101.2C8 by growing the cells in increasing concentrations of the antibody. The resistant cells regained contact inhibition and had a 10-fold lower level of cytoplasmic nucleolin compared to the parental MCF-7 cells. CP101.2C8 is a potent inhibitor of tumor cell viability in vitro. IC50 values of less than 1 μg/ml were obtained for CP101.2C8 versus MV4-11 AML cells, colon, prostate, and lung cancer cells as well as CD33+-CD24- stem cells from MDA-MD-231 breast cancer cells. In contrast, the IC50 concentrations of CP101.2C8 versus normal human B and myeloid cells, breast epithelial cells and lung fibroblasts were greater than 10 μg/ml. Unlike the tumor cells, these normal cells did not express detectable levels of nucleolin in either the plasma membrane or cytoplasm. A fully human recombinant CP101.2C8 is also potent in reducing the viability of MV4-11 cells (IC50 = 0.4 μg/ml). Groups of 10 nu/nu mice bearing SC MV4-11 tumor xenografts were injected IV (every 3 days x 6) with either 10 mg/kg CP101.2C8 or 10 mg/kg of a human IgG1 isotype control antibody. Tumor progression to the 2,000 mm3 endpoint was observed between 24-41 days in all mice treated with the control antibody, while 3/10 of the CP101.2C8-treated mice were long-term survivors that did not reach the endpoint by day 76. The hazard ratio calculated from the Kaplan-Meier plot was 0.22. CP101.2C8 was well tolerated by all the mice; the only adverse event observed was a transient loss in mean body weight of 16%. The widespread and aberrant expression of the multi-functional protein nucleolin in human tumor cells, in contrast to the corresponding normal cells, explains both the broad-spectrum anticancer activity and tumor selectivity of antibody CP101.2C8. Citation Format: Daniel J. Fernandes, Baby G. Tholanikunnel. Development of anti-nucleolin antibodies with broad spectrum anticancer activity and negligible toxicity to normal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1488.

Abstract 4623: Human anti-nucleolin antibodies with broad spectrum anticancer activity

Extensive preclinical studies have validated nucleolin as a new therapeutic target in oncology. Confocal microscopy, immunohistochemistry, and cell fractionation studies have shown that this protein is highly overexpressed in the plasma membrane and cytoplasm of a wide variety of human hematological and solid tumor cells, but is usually undetectable on the cell surface or in the cytoplasm of the corresponding normal cells. We have utilized our licensed platform technology to generate a first-in-class panel of eight fully human monoclonal IgG1 antibodies (HuMAbs). These antibodies bind specifically to nucleolin on the tumor cell surface, which in turn elicits potent cytotoxicity to a variety of human tumor cell lines. One such example is CP101, which killed MV4-11 (AML), MCF-7 (breast), DU145 (prostate), PANC-1 and MIA PaCa-2 (pancreas) tumor cell lines with IC50 values ranging from (0.5-2.0 µg/ml; 3-12 nM) following exposure to the HuMAb for 96 hrs. These in vitro assays were performed in the absence of human complement and immune effector cells required for complement-dependent cellular cytotoxicity (CDCC) and antibody-dependent cellular cytotoxicity (ADCC), respectively. These results are consistent with published observations that anti-nucleolin HuMAbs can exploit the known shuttling function of cell surface nucleolin to gain intracellular access and induce direct tumor cytotoxicity. In contrast to its cytocidal effects on tumor cells, CP101 had no effect on the viability of either MCF-10A normal human breast epithelial cells or normal human CD19+ B cells, neither of which expresses nucleolin on the cell surface. In summary, the results suggest that anti-nucleolin HuMAbs are unique in that they can exert broad spectrum antitumor activity independently of the immune mechanism of CDCC and ADCC, while having no detectable effects on the viability of the corresponding normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4623. doi:1538-7445.AM2012-4623