1L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/b-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IkB-NF-kB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1-ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-kB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-kB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1-ezrin-NF-kB pathway, that induces IGFBP-2 expression, has an important role in CRC progression.Oncogene ( In recent studies, we identified members of the immunoglobulin-like cell adhesion receptors (Nr-CAM and L1), mostly known for their function in nerve cells, 3,4 but also in many cancer cell types, 5 as target genes of b-catenin-TCF signaling in CRC cells. 6,7 We detected L1 in a subpopulation of cells at the invasive front of CRC tissue displaying nuclear b-catenin localization. 7 We further found that the expression of L1 in human CRC cells, lacking endogenous L1, confers enhanced motility and metastasis to the liver in a mouse metastasis model.

Overactivation of Wnt-β-catenin signaling, including β-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a β-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

The transmembrane neural cell adhesion receptor L1 is a Wnt/b-catenin target gene expressed in many tumor types. In human colorectal cancer, L1 localizes preferentially to the invasive front of tumors and when overexpressed in colorectal cancer cells, it facilitates their metastasis to the liver. In this study, we investigated genes that are regulated in human colorectal cancer and by the L1-NF-kB pathway that has been implicated in liver metastasis. c-Kit was the most highly suppressed gene in both colorectal cancer tissue and the L1-NF-kB pathway. c-Kit suppression that resulted from L1-mediated signaling relied upon NF-kB, which directly inhibited the transcription of SP1, a major activator of the c-Kit gene promoter. Reconstituting c-Kit expression in L1-transfected cells blocked the biological effects conferred by L1 overexpression in driving motility and liver metastasis. We found that c-Kit expression in colorectal cancer cells is associated with a more pronounced epithelial morphology, along with increased expression of E-cadherin and decreased expression of Slug. Although c-Kit overexpression inhibited the motility and metastasis of L1-expressing colorectal cancer cells, it enhanced colorectal cancer cell proliferation and tumorigenesis, arguing that separate pathways mediate tumorigenicity and metastasis by c-Kit. Our findings provide insights into how colorectal cancer metastasizes to the liver, the most common site of dissemination in this cancer. Cancer Res; 73(18); 5754-63. Ó2013 AACR.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs–mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression. Implications: PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance. Disclosures No relevant conflicts of interest to declare.