AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced As1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.

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The anticancer AS1411 agent is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. It efficiently inhibits proliferation and induces cell death in many types of cancer cells, but has little effect on normal cells. We have also shown that AS1411 is taken up by macropinocytosis and stimulates further macropinocytosis by a nucleolin-dependent mechanism in several cancer cells. AS1411 activity correlates with stimulated macropinocytosis, suggesting this hyperstimulation of macropinocytosis may explain the unusual cancer cell death caused by AS1411. Macropinocytosis is a ligand-independent endocytic pathway that is normally activated by growth factor receptor stimulation. On the other hand, nucleolin has been reported that can enhance growth factor signaling by binding directly to some growth factor receptors. Therefore in this study, we determine the participation of growth factor signaling pathway in AS1411-induced macropinocytosis and cell death. Pre-incubation of DU145 cells with a specific EGFR inhibitor, but not PDGFR or JAK2 inhibitors, significantly inhibited the stimulation of AS1411-mediated macropinocytosis, suggesting that AS1411 activates EGFR signaling pathways. This was confirmed by observing that DU145 cells treated with AS1411 induced tyrosine phosphorylation of EGFR and activation of its downstream effectors, including p38, PI3K and Rac1. Furthermore, AS1411 seem to induce the interaction between EGFR and nucleolin in accord with EGFR activation. We also found that the activation of EGFR signaling pathway by AS1411 might regulate its cancer cellular response. Simulation of macropinocytosis by AS1411 was significantly inhibited in DU145 cells pre-treated with pharmacological inhibitors of Rac1 or PI3K, but not by Src inhibitor. Moreover the ability of AS1411 to inhibit cell proliferation and induce cell death was reduced by an EGFR-neutralizing antibody (cetuximab) or by downregulating the expression of Rac1 using siRNAs. These results indicate that AS1411 mediates the activation of EGFR signaling pathway to induce cancer cell death, and suggest that AS1411 might redirect EGFR signaling pathway from a cell survival signal to a cell death signal. Citation Format: Elsa M. Reyes-Reyes, Francesca R. Salipur, Paula J. Bates. AS1411 activity is regulated by epidermal growth factor receptor signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2013-1046

XB05 is synthetic small molecule which was serendipitously discovered to have potent anticancer properties. XB05 displays selectivity for malignant cells over non-malignant cells. Human myeloid leukemia cells (U937) and human lung cancer cells (A549) treated with XB05 show an increase in oxidative stress, DNA damage and a mixed apoptotic and necrotic mechanism of cell death. We conducted a COMPARE analysis to search for correlations between XB05 response and gene expression using publicly available microarray studies of the National Cancer Institute 60 cell line panel. This analysis uncovered a correlation between cell death in response to XB05 and expression of the transcription factor, SOX9. We find that SOX9 is differentially expressed across a panel of human lung cancer cell lines. MTT proliferation and clonogenic cell survival assays suggest that SOX9 expression positively correlates with the sensitivity of lung cancer cells to XB05. Furthermore, we show that siRNA knockdown of SOX9 recapitulates the phenotype of XB05-induced cell death, and that SOX9 may be downregulated at the protein level by XB05 treatment. Overall, these data demonstrate potent activity of XB05 against human cancer cells via a novel mechanism of action, and a potential role for SOX9 as a biomarker for selection and monitoring of therapy with XB05. Citation Format: Francesca R. Salipur, Elsa M. Reyes Reyes, Bo Xu, Ned Smith, Jian Cai, Gerald B. Hammond, Paula J. Bates. XB05: a promising small molecule for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2103. doi:10.1158/1538-7445.AM2013-2103