Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/ß-Catenin signaling through Frizzled2-LRP6 assembly.

BACKGROUND: Cancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown. METHODS: The role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis. RESULTS: Ninj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/ß-catenin signaling pathway. CONCLUSIONS: Ninj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/ß-catenin signaling.

A novel C-terminal heat shock protein 90 inhibitor that overcomes STAT3-Wnt-ß-catenin signaling-mediated drug resistance and adverse effects.

Rationale: The heat shock protein (Hsp) system plays important roles in cancer stem cell (CSC) and non-CSC populations. However, limited efficacy due to drug resistance and toxicity are obstacles to clinical use of Hsp90 inhibitors, suggesting the necessity to develop novel Hsp90 inhibitors overcoming these limitations. Methods: The underlying mechanism of resistance to Hsp90 inhibitors was investigated by colony formation assay, sphere formation assay, western blot analysis, and real-time PCR. To develop anticancer Hsp90 inhibitors that overcome the signal transducer and activator of transcription 3 (STAT3)-mediated resistance, we synthesized and screened a series of synthetic deguelin-based compounds in terms of inhibition of colony formation, migration, and viability of non-small cell lung cancer (NSCLC) cells and toxicity to normal cells. Regulation of Hsp90 by the selected compound NCT-80 [5-methoxy-N-(3-methoxy-4-(2-(pyridin-3-yl)ethoxy)phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide] was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. The antitumor, antimetastatic, and anti-CSC effects of NCT-80 were examined in vitro and in vivo using various assays such as MTT, colony formation, and migration assays and flow cytometric analysis and tumor xenograft models. Results: We demonstrated a distinct mechanism in which Hsp90 inhibitors that block N-terminal ATP-binding pocket causes transcriptional upregulation of Wnt ligands through Akt- and ERK-mediated activation of STAT3, resulting in NSCLC cell survival in an autocrine or paracrine manner. In addition, NCT-80 effectively reduced viability, colony formation, migration, and CSC-like phenotypes of NSCLC cells and their sublines with acquired resistance to anticancer drugs by inducing apoptosis and inhibiting epithelial-mesenchymal transition and the growth of NSCLC patient-derived xenograft tumors without overt toxicity. With regards to mechanism, NCT-80 directly bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the interaction between Hsp90 and STAT3 and degrading STAT3 protein. Moreover, NCT-80 inhibited chemotherapy- and EGFR TKI-induced programmed cell death ligand 1 expression and potentiated the antitumor effect of chemotherapy in the LLC-Luc allograft model. Conclusions: These data indicate the potential of STAT3/Wnt signaling pathway as a target to overcome resistance to Hsp90 inhibitors and NCT-80 as a novel Hsp90 inhibitor that targets both CSCs and non-CSCs in NSCLC.

Development of the phenylpyrazolo[3,4-d]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor.

Rationale: The type I insulin-like growth factor receptor (IGF-1R) signaling pathway plays key roles in the development and progression of numerous types of human cancers, and Src and AXL have been found to confer resistance to anti-IGF-1R therapies. Hence, co-targeting Src and AXL may be an effective strategy to overcome resistance to anti-IGF-1R therapies. However, pharmacologic targeting of these three kinases may result in enhanced toxicity. Therefore, the development of novel multitarget anticancer drugs that block IGF-1R, Src, and AXL is urgently needed. Methods: We synthesized a series of phenylpyrazolo[3,4-d]pyrimidine (PP)-based compounds, wherein the PP module was conjugated with 2,4-bis-arylamino-1,3-pyrimidines (I2) via a copper(I)-catalyzed alkyne-azide cycloaddition reaction. To develop IGF-1R/Src/AXL-targeting small molecule kinase inhibitors, we selected LL6 as an active compound and evaluated its antitumor and antimetastatic effects in vitro and in vivo using the MTT assay, colony formation assays, migration assay, flow cytometric analysis, a tumor xenograft model, the KrasG12D/+ -driven spontaneous lung tumorigenesis model, and a spontaneous metastasis model using Lewis lung carcinoma (LLC) allografts. We also determined the toxicity of LL6 in vitro and in vivo. Results: LL6 induced apoptosis and suppressed viability and colony-forming capacities of various non-small cell lung cancer (NSCLC) cell lines and their sublines with drug resistance. LL6 also suppressed the migration of NSCLC cells at nontoxic doses. Administration of LL6 in mice significantly suppressed the growth of NSCLC xenograft tumors and metastasis of LLC allograft tumors with outstanding toxicity profiles. Furthermore, the multiplicity, volume, and load of lung tumors in KrasG12D/+ transgenic mice were substantially reduced by the LL6 treatment. Conclusions: Our results show the potential of LL6 as a novel IGF-1R/Src/AXL-targeting small molecule kinase inhibitor, providing a new avenue for anticancer therapies.

RGS2-mediated translational control mediates cancer cell dormancy and tumor relapse.

Slow-cycling/dormant cancer cells (SCCs) have pivotal roles in driving cancer relapse and drug resistance. A mechanistic explanation for cancer cell dormancy and therapeutic strategies targeting SCCs are necessary to improve patient prognosis, but are limited because of technical challenges to obtaining SCCs. Here, by applying proliferation-sensitive dyes and chemotherapeutics to non-small cell lung cancer (NSCLC) cell lines and patient-derived xenografts, we identified a distinct SCC subpopulation that resembled SCCs in patient tumors. These SCCs displayed major dormancy-like phenotypes and high survival capacity under hostile microenvironments through transcriptional upregulation of regulator of G protein signaling 2 (RGS2). Database analysis revealed RGS2 as a biomarker of retarded proliferation and poor prognosis in NSCLC. We showed that RGS2 caused prolonged translational arrest in SCCs through persistent eukaryotic initiation factor 2 (eIF2α) phosphorylation via proteasome-mediated degradation of activating transcription factor 4 (ATF4). Translational activation through RGS2 antagonism or the use of phosphodiesterase 5 inhibitors, including sildenafil (Viagra), promoted ER stress-induced apoptosis in SCCs in vitro and in vivo under stressed conditions, such as those induced by chemotherapy. Our results suggest that a low-dose chemotherapy and translation-instigating pharmacological intervention in combination is an effective strategy to prevent tumor progression in NSCLC patients after rigorous chemotherapy.

Evodiamine inhibits both stem cell and non-stem-cell populations in human cancer cells by targeting heat shock protein 70.

Rationale: Cancer stem cells (CSCs) are known to cause tumor recurrence and drug resistance. The heat shock protein (HSP) system plays a major role in preserving expression and function of numerous oncoproteins, including those involved in the CSC activities. We explored novel anticancer drugs, especially those targeting HSP components required for the functional role of CSCs. Methods: Investigation of the role of the HSP system in CSCs and screening of a natural product chemical library were performed by utilizing cancer cell lines, primary cultures of patient-derived xenografts (PDXs), and their putative CSC subpopulations (i.e., those grown under sphere-forming conditions, stably transfected with reporter vectors carrying NANOG or POUSF1 promoters, or carrying high ALDH activity) in vitro and PDX and KrasG12D/+-driven tumor models in vivo. Regulation of the HSP system was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. Results: The HSP system was activated in CSCs via transcriptional upregulation of the HSP system components, especially HSP70. Evodiamine (Evo) was identified to induce apoptosis in both CSC and bulk non-CSC populations in human lung, colon, and breast cancer cells and their sublines with chemoresistance. Evo administration decreased the multiplicity, volume, and load of lung tumors in KrasG12D/+ transgenic mice and the growth of cancer cell line- and PDX-derived tumors without detectable toxicity. Mechanistically, Evo disrupted the HSP system by binding the N-terminal ATP-binding pocket of HSP70 and causing its ubiquitin-mediated degradation. Conclusions: Our findings illustrate HSP70 as a potential target for eliminating CSCs and Evo as an effective HSP70-targeting anticancer drug eradicating both CSCs and non-CSCs with a minimal toxicity.

Potent Anticancer Effect of the Natural Steroidal Saponin Gracillin Is Produced by Inhibiting Glycolysis and Oxidative Phosphorylation-Mediated Bioenergetics.

Metabolic rewiring to utilize aerobic glycolysis is a hallmark of cancer. However, recent findings suggest the role of mitochondria in energy generation in cancer cells and the metabolic switch to oxidative phosphorylation (OXPHOS) in response to the blockade of glycolysis. We previously demonstrated that the antitumor effect of gracillin occurs through the inhibition of mitochondrial complex II-mediated energy production. Here, we investigated the potential of gracillin as an anticancer agent targeting both glycolysis and OXPHOS in breast and lung cancer cells. Along with the reduction in adenosine triphosphate (ATP) production, gracillin markedly suppresses the production of several glycolysis-associated metabolites. A docking analysis and enzyme assay suggested phosphoglycerate kinase 1 (PGK1) is a potential target for the antiglycolytic effect of gracillin. Gracillin reduced the viability and colony formation ability of breast cancer cells by inducing apoptosis. Gracillin displayed efficacious antitumor effects in mice bearing breast cancer cell line or breast cancer patient-derived tumor xenografts with no overt changes in body weight. An analysis of publicly available datasets further suggested that PGK1 expression is associated with metastasis status and poor prognosis in patients with breast cancer. These results suggest that gracillin is a natural anticancer agent that inhibits both glycolysis and mitochondria-mediated bioenergetics.

The Interplay between Slow-Cycling, Chemoresistant Cancer Cells and Fibroblasts Creates a Proinflammatory Niche for Tumor Progression.

Quiescent cancer cells are believed to cause cancer progression after chemotherapy through unknown mechanisms. We show here that human non-small cell lung cancer (NSCLC) cell line-derived, quiescent-like, slow-cycling cancer cells (SCC) and residual patient-derived xenograft (PDX) tumors after chemotherapy experience activating transcription factor 6 (ATF6)-mediated upregulation of various cytokines, which acts in a paracrine manner to recruit fibroblasts. Cancer-associated fibroblasts (CAF) underwent transcriptional upregulation of COX2 and type I collagen (Col-I), which subsequently triggered a slow-to-active cycling switch in SCC through prostaglandin E2 (PGE2)- and integrin/Src-mediated signaling pathways, leading to cancer progression. Both antagonism of ATF6 and cotargeting of Src/COX2 effectively suppressed cytokine production and slow-to-active cell cycling transition in SCC, withholding cancer progression. Expression of COX2 and Col-I and activation of Src were observed in patients with NSCLC who progressed while receiving chemotherapy. Public data analysis revealed significant association between COL1A1 and SRC expression and NSCLC relapse. Overall, these findings indicate that a proinflammatory niche created by the interplay between SCC and CAF triggers tumor progression. SIGNIFICANCE: Cotargeting COX2 and Src may be an effective strategy to prevent cancer progression after chemotherapy.

The natural compound gracillin exerts potent antitumor activity by targeting mitochondrial complex II.

Mitochondria play a pivotal role in cancer bioenergetics and are considered a potential target for anticancer therapy. Considering the limited efficacy and toxicity of currently available mitochondria-targeting agents, it is necessary to develop effective mitochondria-targeting anticancer drugs. By screening a large chemical library consisting of natural products with diverse chemical entities, we identified gracillin, a steroidal saponin, as a mitochondria-targeting antitumor drug. Gracillin displayed broad-spectrum inhibitory effects on the viability of a large panel of human cancer cell lines, including those carrying acquired resistance to chemotherapy or EGFR-targeting drugs, by inducing apoptosis. We show that gracillin attenuates mitochondria-mediated cellular bioenergetics by suppressing ATP synthesis and by producing reactive oxygen species (ROS). Mechanistically, gracillin disrupts complex II (CII) function by abrogating succinate dehydrogenase (SDH) activity without affecting the succinate:ubiquinone reductase. The gracillin-induced cell death was potentiated by 3-nitropropionic acid (3-NPA) or thenoyltrifluoroacetone (TTFA), which inhibit CII by binding to the active site of SDHA or to the ubiquinone-binding site, respectively. Finally, we show that gracillin effectively suppressed the mutant-Kras-driven lung tumorigenesis and the growth of xenograft tumors derived from cell lines or patient tissues. Gracillin displayed no obvious pathophysiological features in mice. Collectively, gracillin has potential as a CII-targeting antitumor drug.

Development of a novel Hsp90 inhibitor NCT-50 as a potential anticancer agent for the treatment of non-small cell lung cancer.

Despite the development of advanced therapeutic regimens such as molecular targeted therapy and immunotherapy, the 5-year survival of patients with lung cancer is still less than 20%, suggesting the need to develop additional treatment strategies. The molecular chaperone heat shock protein 90 (Hsp90) plays important roles in the maturation of oncogenic proteins and thus has been considered as an anticancer therapeutic target. Here we show the efficacy and biological mechanism of a Hsp90 inhibitor NCT-50, a novobiocin-deguelin analog hybridizing the pharmacophores of these known Hsp90 inhibitors. NCT-50 exhibited significant inhibitory effects on the viability and colony formation of non-small cell lung cancer (NSCLC) cells and those carrying resistance to chemotherapy. In contrast, NCT-50 showed minimal effects on the viability of normal cells. NCT-50 induced apoptosis in NSCLC cells, inhibited the expression and activity of several Hsp90 clients including hypoxia-inducible factor (HIF)-1α, and suppressed pro-angiogenic effects of NSCLC cells. Further biochemical and in silico studies revealed that NCT-50 downregulated Hsp90 function by interacting with the C-terminal ATP-binding pocket of Hsp90, leading to decrease in the interaction with Hsp90 client proteins. These results suggest the potential of NCT-50 as an anticancer Hsp90 inhibitor.

Development of a 4-aminopyrazolo[3,4-d]pyrimidine-based dual IGF1R/Src inhibitor as a novel anticancer agent with minimal toxicity.

BACKGROUND: Both the type I insulin-like growth factor receptor (IGF1R) and Src pathways are associated with the development and progression of numerous types of human cancer, and Src activation confers resistance to anti-IGF1R therapies. Hence, targeting both IGF1R and Src concurrently is one of the main challenges in combating resistance to the currently available anti-IGF1R-based anticancer therapies. However, the enhanced toxicity from this combinatorial treatment could be one of the main hurdles for this strategy, suggesting the necessity of developing a novel strategy for co-targeting IGF1R and Src to meet an urgent clinical need. METHODS: We synthesized a series of 4-aminopyrazolo[3,4-d]pyrimidine-based dual IGF1R/Src inhibitors, selected LL28 as an active compound and evaluated its potential antitumor effects in vitro and in vivo using the MTT assay, colony formation assays, flow cytometric analysis, a tumor xenograft model, and the Kras G12D/+ -driven spontaneous lung tumorigenesis model. RESULTS: LL28 markedly suppressed the activation of IGF1R and Src and significantly inhibited the viability of several NSCLC cell lines in vitro by inducing apoptosis. Administration of mice with LL28 significantly suppressed the growth of H1299 NSCLC xenograft tumors without overt toxicity and substantially reduced the multiplicity, volume, and load of lung tumors in the Kras G12D/+ -driven lung tumorigenesis model. CONCLUSIONS: The present results suggest the potential of LL28 as a novel anticancer drug candidate targeting both IGF1R and Src, providing a new avenue to efficient anticancer therapies. Further investigation is warranted in advanced preclinical and clinical settings.

Panaxynol, a natural Hsp90 inhibitor, effectively targets both lung cancer stem and non-stem cells.

Cancer stem-like cells (CSCs) contribute to tumor recurrence and chemoresistance. Hence, strategies targeting CSCs are crucial for effective anticancer therapies. Here, we demonstrate the capacities of the non-saponin fraction of Panax ginseng and its active principle panaxynol to inhibit Hsp90 function and viability of both non-CSC and CSC populations of NSCLC in vitro and in vivo. Panaxynol inhibited the sphere forming ability of NSCLC CSCs at nanomolar concentrations, and micromolar concentrations of panaxynol suppressed the viability of NSCLC cells (non-CSCs) and their sublines carrying acquired chemoresistance with minimal effect on normal cells derived from various organs. Orally administered panaxynol significantly reduced lung tumorigenesis in KrasG12D/+ transgenic mice and mice carrying NSCLC xenografts without detectable toxicity. Mechanistically, panaxynol disrupted Hsp90 function by binding to the N-terminal and C-terminal ATP-binding pockets of Hsp90 without increasing Hsp70 expression. These data suggest the potential of panaxynol as a natural Hsp90 inhibitor targeting both the N-terminal and C-terminal of Hsp90 with limited toxicities.

Essential Role of DNA Methyltransferase 1-mediated Transcription of Insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors.

Purpose: Histone deacetylase inhibitors (HDI) are promising anticancer therapies; however, drug resistance limits their efficacy. Here, we investigated the molecular mechanisms underlying HDI resistance, focusing on the mechanism of HDI-mediated induction of insulin-like growth factor 2 (IGF2) based on our previous study.Experimental Design: The methylation status of CCCTC-binding factor (CTCF)-binding sites in the IGF2/H19 imprinting control region (ICR) were determined by methylation-specific PCR and bisulfite sequencing. The effectiveness of single or combinatorial blockade of DNA methyltransferase 1 (DNMT1) and histone deacetylase (HDAC) was evaluated using cell viability assay and patient-derived tumor xenograft (PDX) model.Results: HDAC inhibition by vorinostat increased acetylated STAT3 (K685), resulting in transcriptional upregulation of DNMT1 DNMT1-mediated hypermethylation of CTCF-binding sites in the IGF2/H19 ICR decreased CTCF insulator activity, leading to a transcriptional upregulation of IGF2 and activation of the insulin-like growth factor 1 receptor (IGF-1R) pathway in cells with acquired or de novo vorinostat resistance. Strategies targeting DNMT1 diminished the IGF2 expression and potentiated vorinostat sensitivity in preclinical models of lung cancer with hypermethylation in the H19/IGF2 ICR. The degree of ICR hypermethylation correlated with vorinostat resistance in patient-derived lung tumors and in patients with hematologic malignancies.Conclusions: DNMT1-mediated transcriptional upregulation of IGF2 is a novel mechanism of resistance to HDIs, highlighting the role of epigenetic deregulation of IGF2 in HDI resistance and the potential value of the H19/IGF2 ICR hypermethylation and DNMT1 expression as predictive biomarkers in HDI-based anticancer therapies. Clin Cancer Res; 23(5); 1299-311. ©2016 AACR.

Deguelin Analogue SH-1242 Inhibits Hsp90 Activity and Exerts Potent Anticancer Efficacy with Limited Neurotoxicity.

The Hsp90 facilitates proper folding of signaling proteins associated with cancer progression, gaining attention as a target for therapeutic intervention. The natural rotenoid deguelin was identified as an Hsp90 inhibitor, but concerns about neurotoxicity have limited prospects for clinical development. In this study, we report progress on deguelin analogues that address this limitation, focusing on the novel analogue SH-1242 as a candidate to broadly target human lung cancer cells, including those that are chemoresistant or harboring KRAS mutations. In a KRAS-driven mouse model of lung cancer, SH-1242 administration reduced tumor multiplicity, volume, and load. Similarly, in human cell line-based or patient-derived tumor xenograft models, SH-1242 induced apoptosis and reduced tumor vasculature in the absence of detectable toxicity. In contrast to deguelin, SH-1242 toxicity was greatly reduced in normal cells and when administered to rats did not produce obvious histopathologic features in the brain. Mechanistic studies revealed that SH-1242 bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the ability to interact with its co-chaperones and clients and triggering a degradation of client proteins without affecting Hsp70 expression. Taken together, our findings illustrate the superior properties of SH-1242 as an Hsp90 inhibitor and as an effective antitumor and minimally toxic agent, providing a foundation for advancing further preclinical and clinical studies.

Activation of insulin-like growth factor receptor signaling mediates resistance to histone deacetylase inhibitors.

Histone deacetylases (HDACs) are considered promising targets in the treatment of hematologic malignancies and several types of solid tumors, including non-small cell lung cancer (NSCLC). However, the efficacy of HDAC inhibitors in solid tumors is marginal, and the mechanisms underlying resistance to HDAC inhibitors are largely unknown. Here, we demonstrate the involvement of type 1 insulin-like growth factor receptor (IGF-1R) signaling in resistance to HDAC inhibitors in NSCLC. Using MTT and soft-agar colony formation assays, we selected NSCLC cell lines that exhibited intrinsic resistance to vorinostat. Treatment with vorinostat activated IGF-1R signaling in vorinostat-resistant but not vorinostat-sensitive NSCLC cells. Other HDAC inhibitors, including trichostatin A, sodium butyrate, and depsipeptide, also activated IGF-1R signaling in vorinostat-resistant NSCLC cells. Blockade of IGF-1R signaling via IGF-1R monoclonal antibodies (mAbs) or through knockdown of IGF-1R via RNA interference sensitized vorinostat-resistant cells to HDAC inhibition. Finally, IGF-1R mAbs sensitized xenograft tumors of vorinostat-resistant cells to vorinostat treatment in vivo. These findings suggest that IGF-1R activation is generally involved in resistance to HDAC inhibitors and that targeting IGF-1R is an effective strategy for overcoming resistance to HDAC inhibitors in NSCLC.