The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.

Human anaplastic lymphoma kinase (ALK) has been identified as an oncogene that is mutated or amplified in NBLs. To obtain a better understanding of the molecular events associated with ALK in the pathogenesis of NBL, it is necessary to clarify how ALK gene contributes to NBL progression. In the present study, we found that ALK expression was significantly high in NBL clinical samples with amplified MYCN (n = 126, P < 0.01) and in developing tumors of MYCN-transgenic mice. Indeed, promoter analysis revealed that ALK is a direct transcriptional target of MYCN. Overexpression and knockdown of ALK demonstrated its function in cell proliferation, migration and invasion. Moreover, treatment with an ALK inhibitor, TAE-684, efficiently suppressed such biological effects in MYCN amplified cells and tumor growth of the xenograft in mice. Our present findings explore the fundamental understanding of ALK in order to develop novel therapeutic tools by targeting ALK for aggressive NBL treatment.

The mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1) mediates activin B signals required for eyelid epithelium morphogenesis during mouse fetal development. The present study investigates the role of MEKK1 in epithelial wound healing, another activin-regulated biological process. In a skin wound model, injury markedly stimulates MEKK1 expression and activity, which are in turn required for the expression of genes involved in extracellular matrix (ECM) homeostasis. MEKK1 ablation or down-regulation by interfering RNA significantly delays skin wound closure and impairs activation of Jun NH 2 -terminal kinases, induction of plasminogen activator inhibitor (PAI)-1, and restoration of cell-cell junctions of the wounded epidermis. Conversely, expression of wild-type MEKK1 accelerates reepithelialization of full-thickness skin and corneal debridement wounds by mechanisms involving epithelial cell migration, a cell function that is partially abolished by neutralizing antibodies for PAI-1 and metalloproteinase III. Our data suggest that MEKK1 transmits wound signals, leading to the transcriptional activation of genes involved in ECM homeostasis, epithelial cell migration, and wound reepithelialization.

Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1 . MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure.

*JNK1 and JNK2 are two ubiquitously expressed isoforms that exert redundant roles in many physiological processes, but the extent of their relative contributions to these processes has not been well characterized. We show that both JNK isoforms transmit MEK kinase 1 (MEKK1)-mediated morphogenetic signals during mouse embryonic eyelid closure. However, JNK1 and JNK2 are not synonymous, because MEKK1 is haploinsufficient for normal eyelid closure in Jnk1-null mice, but is haplosufficient in Jnk2-null mice. In the Mekk1 heterozygous background, a more efficient phosphorylation of JNK1 than JNK2 leads to differential downstream reactions, such as c-Jun phosphorylation and PAI1 expression in the developing eyelid epithelium. Differences in efficiency of phosphorylation are attributed to JNK1 Gly177 and Ser179 -residues that are absent in JNK2 -which promote a less ordered structural conformation. This leads to more favorable JNK phosphorylation by activin B morphogenetic signals mediated by the MEKK1-MKK4 pathway. Interestingly, Mekk1-Jnk1-Jnk2 triple hemizygotes display a partial eye-open phenotype at birth, suggesting that all three genes dose-dependently contribute to morphogenetic eyelid closure. We propose that a MEKK1-JNK1/2 axis governs the JNK activation levels to control downstream transcriptional events and eyelid morphogenesis and that reduction of upstream MEKK1 signals uncovers analogous but differential roles of JNK1 and JNK2 in a biological process.

Neuronal leucine-rich repeat protein-1 (NLRR1), a type-1 transmembrane protein highly expressed in unfavorable neuroblastoma, is a target gene of MYCN that is predominately expressed in primary neuroblastomas with MYCN amplification. However, the precise biological role of NLRR1 in cell proliferation and tumor progression remains unknown. To investigate its functional importance, we examined the role of NLRR1 in EGF and insulin growth factor-1 (IGF-1)-mediated cell viability. We found that NLRR1 positively regulated cell proliferation through activation of extracellular signal-regulated kinase mediated by EGF and IGF-1. Interestingly, EGF stimulation induced endogenous MYCN expression through Sp1 recruitment to the MYCN promoter region, which was accelerated in NLRR1-expressing cells. The Sp1-binding site was identified on the promoter region for MYCN induction, and phosphorylation of Sp1 was important for EGF-mediated MYCN regulation. In vivo studies confirmed the proliferation-promoting activity of NLRR1 and established an association between NLRR1 expression and poor prognosis in neuroblastoma. Together, our findings indicate that NLRR1 plays an important role in the development of neuroblastoma and therefore may represent an attractive therapeutic target for cancer treatment. Cancer Res; 72(17); 4587-96. Ó2012 AACR.

Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein inspired us to develop a new gene-level-inhibitory strategy using a sequence-specific gene regulator. Here, we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within the MYCN transcript. Pharmacologic suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN nonamplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expres-sion and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, treatment with MYCN-A3, but not MYCN nontargeting PI polyamide, precipitated a copy number reduction of MYCN in neuroblastoma cells with MYCN amplification. These findings suggest that directly targeting MYCN with MYCN-A3 is a novel therapeutic approach to reduce copy number of the MYCN gene for MYCN-amplified neuroblastoma.Significance: This study presents a novel approach to drugging an amplified oncogene by showing that targeting gene amplification of MYCN suppresses MYCN expression and neuroblastoma growth.