David J. Feith

Network modeling of TGFβ signaling in hepatocellular carcinoma epithelial-to-mesenchymal transition reveals joint Sonic hedgehog and Wnt pathway activation

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC.

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

Ornithine decarboxylase is a target for chemoprevention of basal and squamous cell carcinomas in Ptch1+/– mice

Solar ultraviolet B (UVB) radiation induces cutaneous ornithine decarboxylase (ODC), the first enzyme in the polyamine-biosynthesis pathway, which drives continued proliferation and clonal expansion of initiated (mutated) cells, leading to tumorigenesis. Therefore ODC is a potentially important target for chemoprevention of basal cell carcinomas (BCCs), the majority of which have mutations in the tumor-suppressor gene known as patched (PTCH). To assess this possibility, we first overexpressed ODC in the skin of Ptch1+/- mice using a keratin 6 (K6) promoter that directs constitutive ODC expression in the outer root sheath of the hair follicle. UVB irradiation of these mice accelerated induction of BCCs as compared with their Ptch1+/- littermates. To further verify the role of ODC in BCC tumorigenesis, we used an antizyme (AZ) approach to inhibit ODC activity in the Ptch1+/- mice. Ptch1+/- mice with AZ overexpression driven by the K6 promoter were resistant to the induction of BCCs by UVB. Furthermore, oral administration of the suicidal ODC inhibitor alpha-difluoromethylornithine reduced UVB-induced BCCs in Ptch1+/- mice. These results demonstrate the crucial importance of ODC for the induction of BCCs and indicate that chemopreventive strategies directed at inhibiting this enzyme may be useful in reducing BCCs in human populations.

Polyamines and neoplastic growth

Studies over many years have suggested that increased polyamine synthesis may be necessary for neoplastic growth. This review summarizes recent work on the regulation of putrescine production both de novo and via the degradation of higher polyamines and provides a summary of studies using transgenic mice in which the levels of proteins that regulate these processes (L-ornithine decarboxylase, antizyme and spermidine/spermine-N(1)-acetyltransferase) are altered.

Targeted overexpression of ornithine decarboxylase enhances beta-adrenergic agonist-induced cardiac hypertrophy.

These studies were designed to determine the consequences of constitutive overexpression of ornithine decarboxylase (ODC) in the heart. Induction of ODC is known to occur in response to agents that induce cardiac hypertrophy. However, it is not known whether high ODC levels are sufficient for the development of a hypertrophic phenotype. Transgenic mice were generated with cardiac-specific expression of a stable ODC protein using the alpha-myosin heavy-chain promoter. Founder lines with >1000-fold overexpression of ODC in the heart were established, resulting in a 50-fold overaccumulation of putrescine, 4-fold elevation in spermidine, a slight increase in spermine and accumulation of large amounts of cadaverine compared with littermate controls. Despite these significant alterations in polyamines, myocardial hypertrophy, as measured by ratio of heart to body weight, did not develop, although atrial natriuretic factor RNA was slightly elevated in transgenic ventricles. However, stimulation of beta-adrenergic signalling by isoproterenol resulted in severe hypertrophy and even death in ODC-overexpressing mice without further altering polyamine levels, compared with only a mild hypertrophy in littermates. When beta1-adrenergic stimulation was blocked by simultaneous treatment with isoproterenol and the beta1 antagonist atenolol, a significant, although reduced, hypertrophy was still present in the hearts of transgenic mice, suggesting that both beta1 and beta2 adrenergic receptors contribute to the hypertrophic phenotype. Therefore these mice provide a model to study the in vivo co-operativity between high ODC activity and activation of other pathways leading to hypertrophy in the heart.

Novel off-target effect of tamoxifen — Inhibition of acid ceramidase activity in cancer cells

Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5min, and dose-dependent, as low as 5μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism.

C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia.

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.

DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma

Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G1 cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27Kip1 protein and causes p27Kip1/Rb-coupled G1 cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27Kip1 phosphorylation at residues Ser10 and Thr198. While p27Kip1 is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27Kip1 as a regulator of cell migration and invasion. In the present study we found that p27Kip1 regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27Kip1. DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27Kip1 mRNA correlates with clinical features and the survival probability of NB patients.

Combinatorial interventions inhibit TGFβ-driven epithelial-to-mesenchymal transition and support hybrid cellular phenotypes

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is transforming growth factor beta (TGFβ), which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We aim to identify network perturbations that suppress TGFβ-driven EMT, with the goal of suppressing invasive properties of cancer cells. We use a systems-level Boolean dynamic model of EMT to systematically screen individual and combination perturbations (inhibition or constitutive activation of up to four nodes). We use a recently developed network control approach to understand the mechanism through which the combinatorial interventions suppress EMT. We test the results of our in silico analysis using siRNA. Our model predicts that targeting key elements of feedback loops in combination with the SMAD complex is more effective than suppressing the SMAD complex alone. We demonstrate experimentally that expression of a majority of these elements is enriched in mesenchymal relative to epithelial phenotype HCC cell lines. An siRNA screen of the predicted combinations confirms that many targeting strategies suppress TGFβ-driven EMT measured by E-cadherin expression and cell migration. Our analysis reveals that some perturbations give rise to hybrid states intermediate to the epithelial and mesenchymal states. Our results indicate that EMT is driven by an interconnected signaling network and many apparently successful single interventions may lead to steady states that are in-between epithelial and mesenchymal states. As these putative hybrid or partial EMT states may retain invasive properties, our results suggest that combinatorial therapies are necessary to fully suppress invasive properties of tumor cells.

Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy.

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac alpha-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10-39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30-32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.