ALK upregulates POSTN and WNT signaling to drive neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling.

Use of DNA-alkylating pyrrole-imidazole polyamides for anti-cancer drug sensitivity screening in pancreatic ductal adenocarcinoma.

BACKGROUND: Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. METHODS: KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. RESULT: KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. CONCLUSION: These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.

Targeting anaplastic lymphoma kinase (ALK) gene alterations in neuroblastoma by using alkylating pyrrole-imidazole polyamides.

Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.

CD133 prevents colon cancer cell death induced by serum deprivation through activation of Akt-mediated protein synthesis and inhibition of apoptosis.

During the early phase of tumorigenesis, primary malignant cells survive within a low nutrition environment caused by a poorly organized vascular system. Here, we sought to determine the functional significance of CD133 in the survival of cancer cells under nutrient-poor conditions. Knockdown and overexpression experiments demonstrated that CD133 suppresses colon cancer cell death induced by serum deprivation through activation of Akt-mediated anti-apoptosis and protein synthesis pathways. Furthermore, serum deprivation increased the amount of endogenous CD133 protein, which was regulated at least in part by phosphoinositide 3-kinase. Thus, it is highly likely that CD133 contributes to the acquisition/maintenance of the resistance to stress arising from nutrient deficiency in early avascular tumor tissues.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

Vitamin K3 derivative induces apoptotic cell death in neuroblastoma via downregulation of MYCN expression.

Neuroblastoma is a pediatric malignant tumor arising from the sympathetic nervous system. The patients with high-risk neuroblastomas frequently exhibit amplification and high expression of the MYCN gene, resulting in worse clinical outcomes. Vitamin K3 (VK3) is a synthetic VK-like compound that has been known to have antitumor activity against various types of cancers. In the present study, we have asked whether VK3 and its derivative, VK3-OH, could have the antitumor activity against neuroblastoma-derived cells. Based on our results, VK3-OH strongly inhibited cell proliferation and induced apoptotic cell death compared to VK3. Treatment of MYCN-driven neuroblastoma cells with VK3-OH potentiated tumor suppressor p53 accompanied by downregulation of anti-apoptotic Bcl-2 and Mcl-1. Interestingly, VK3-OH also suppressed the MYCN at mRNA and protein levels. Furthermore, we found downregulation of LIN28B following VK3-OH treatment in MYCN-amplified and overexpressed neuroblastoma cells. Collectively, our current findings strongly suggest that VK3-OH provides a potential therapeutic strategy for patients with MYCN-driven neuroblastomas.

PTPRK suppresses progression and chemo-resistance of colon cancer cells via direct inhibition of pro-oncogenic CD133.

Receptor-type protein tyrosine phosphatase κ (PTPRK) is considered to be a candidate tumor suppressor. PTPRK dephosphorylates CD133, which is a stem cell marker; phosphorylated CD133 accelerates xenograft tumor growth of colon cancer cells through the activation of AKT, but the functional significance of this has remained elusive. In this study, we have demonstrated that knockdown of PTPRK potentiates the pro-oncogenic CD133-AKT pathway in colon cancer cells. Intriguingly, depletion of PTPRK significantly reduced sensitivity to the anti-cancer drug oxaliplatin and was accompanied by up-regulation of phosphorylation of Bad, a downstream target of AKT. Together, our present observations strongly suggest that the CD133-PTPRK axis plays a pivotal role in the regulation of colon cancer progression as well as drug resistance.

Estimating genome-wide off-target effects for pyrrole-imidazole polyamide binding by a pathway-based expression profiling approach.

In the search for new pharmaceutical leads, especially with DNA-binding molecules or genome editing methods, the issue of side and off-target effects have always been thorny in nature. A particular case is the investigation into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders with strong affinity to the minor-groove and sequence specificity, but at < 20 bases, their relatively short motifs also insinuate the possibility of non-unique genomic binding. Binding at non-intended loci potentially lead to the rise of off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method to infer off-target binding, via expression profiling, based on probing the relative impact to various biochemical pathways; we also proposed an accompanying side effect prediction engine for the systematic screening of candidate polyamides. This method marks the first attempt in PI polyamide research to identify elements in biochemical pathways that are sensitive to the treatment of a candidate polyamide as an approach to infer possible off-target effects. Expression changes were then considered to assess possible outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We validated some of these effects with a series of animal experiments, and found agreeable corroboration in certain side effects, such as changes in aspartate transaminase levels in ICR and nude mice post-administration.

Direct Targeting of MYCN Gene Amplification by Site-Specific DNA Alkylation in Neuroblastoma.

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 expression 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.

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63.

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate.

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.

Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity.

Although runt-related transcription factor 2 (RUNX2) is known to be an essential key transcription factor for osteoblast differentiation and bone formation, RUNX2 also plays a pivotal role in the regulation of p53-dependent DNA damage response. In the present study, we report that, in addition to p53, RUNX2 downregulates pro-apoptotic TAp73 during DNA damage-dependent cell death. Upon adriamycin (ADR) exposure, human osteosarcoma-derived U2OS cells underwent cell death in association with an upregulation of TAp73 and various p53/TAp73-target gene products together with RUNX2. Small interfering RNA-mediated silencing of p73 resulted in a marked reduction in ADR-induced p53/TAp73-target gene expression, suggesting that TAp73 is responsible for the ADR-dependent DNA damage response. Immunoprecipitation and transient transfection experiments demonstrated that RUNX2 forms a complex with TAp73 and impairs its transcriptional activity. Notably, knockdown of RUNX2 stimulated ADR-induced cell death accompanied by a massive induction of TAp73 expression, indicating that RUNX2 downregulates TAp73 expression. Consistent with this notion, the overexpression of RUNX2 suppressed ADR-dependent cell death, which was associated with a remarkable downregulation of TAp73 and p53/TAp73-target gene expression. Collectively, our present findings strongly suggest that RUNX2 attenuates the transcriptional activity and ADR-mediated induction of TAp73, and may provide novel insights into understanding the molecular basis behind the development and/or maintenance of chemoresistance. Thus, we propose that the silencing of RUNX2 might be an attractive strategy for improving the chemosensitivity of malignant cancers.

Vitamin K3 analogs induce selective tumor cytotoxicity in neuroblastoma.

We investigated the cytotoxicity of eight vitamin K3 (VK3) analogs against neuroblastoma cell lines (IMR-32, LA-N-1, NB-39, and SK-N-SH) and normal cell lines (human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF)) using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. 2-[(2-Methoxy)ethylthio]-3-methyl-1,4-naphthoquinone (VK3-OCH(3)) showed especially potent cytotoxic activities against neuroblastoma cells compared with normal cells. In a Hoechst 33342 staining experiment, apoptotic morphologies characterized by cell shrinkage, nuclear condensation, and nuclear fragmentation were observed in IMR-32 and LA-N-1 cells after 48 h of treatment with 10(-5) M of VK3-OCH(3). To clarify the molecular mechanisms of apoptosis induced by VK3-OCH(3), we examined the expression of apoptosis related proteins using a Proteome Profiler Array and western blotting. Heme oxygenase (HO)-1 was remarkably increased by VK3-OCH(3) compared with the control (173% in IMR-32 and 170% in LA-N-1 at 24 h). Moreover, caveolin-1 was induced by VK3-OCH(3) at 48 h. In addition, VK3-OCH(3) arrested the cell cycle at the G2/M phase in IMR-32 cells. These results suggest that VK3-OCH(3) exhibited a selective antitumor activity via HO-1-related mechanisms.