Mathematical modeling links Wnt signaling to emergent patterns of metabolism in colon cancer

Cell‐intrinsic metabolic reprogramming is a hallmark of cancer that provides anabolic support to cell proliferation. How reprogramming influences tumor heterogeneity or drug sensitivities is not well understood. Here, we report a self‐organizing spatial pattern of glycolysis in xenograft colon tumors where pyruvate dehydrogenase kinase (PDK1), a negative regulator of oxidative phosphorylation, is highly active in clusters of cells arranged in a spotted array. To understand this pattern, we developed a reaction–diffusion model that incorporates Wnt signaling, a pathway known to upregulate PDK1 and Warburg metabolism. Partial interference with Wnt alters the size and intensity of the spotted pattern in tumors and in the model. The model predicts that Wnt inhibition should trigger an increase in proteins that enhance the range of Wnt ligand diffusion. Not only was this prediction validated in xenograft tumors but similar patterns also emerge in radiochemotherapy‐treated colorectal cancer. The model also predicts that inhibitors that target glycolysis or Wnt signaling in combination should synergize and be more effective than each treatment individually. We validated this prediction in 3D colon tumor spheroids.     

PDK4 Protein Promotes Tumorigenesis through Activation of cAMP-response Element-binding Protein (CREB)-Ras Homolog Enriched in Brain (RHEB)-mTORC1 Signaling Cascade

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.     

Wnt meets Warburg: another piece in the puzzle?

One of the most common abnormalities of cancer cells is their predilection to engage in a high rate of glycolysis despite the continued availability of oxygen. First described by Otto Warburg, this phenomenon of aerobic glycolysis (or the Warburg effect) has recently been proposed to result from cancer‐associated alterations in signal transduction pathways. In this issue of The EMBO Journal, Pate et al provide further support for this hypothesis by demonstrating that Wnt signaling plays an important role in establishing aerobic glycolysis as a mechanism to support in vivo cancer cell proliferation.     

Glucose oxidation modulates anoikis and tumor metastasis

Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.     

Pyruvate dehydrogenase kinase 1 contributes to cisplatin resistance of ovarian cancer through EGFR activation

Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial–mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.     

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis

Cancer cells display high rates of aerobic glycolysis, a phenomenon known historically as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are highly produced by cancer cells even in the presence of oxygen. Hypoxia-induced gene expression in cancer cells has been linked to malignant transformation. Here we provide evidence that lactate and pyruvate regulate hypoxia-inducible gene expression independently of hypoxia by stimulating the accumulation of hypoxia-inducible Factor 1alpha (HIF-1alpha). In human gliomas and other cancer cell lines, the accumulation of HIF-1alpha protein under aerobic conditions requires the metabolism of glucose to pyruvate that prevents the aerobic degradation of HIF-1alpha protein, activates HIF-1 DNA binding activity, and enhances the expression of several HIF-1-activated genes including erythropoietin, vascular endothelial growth factor, glucose transporter 3, and aldolase A. Our findings support a novel role for pyruvate in metabolic signaling and suggest a mechanism by which high rates of aerobic glycolysis can promote the malignant transformation and survival of cancer cells.     

lncRNA PCAT19 promotes the proliferation of laryngocarcinoma cells via modulation of the miR-182/PDK4 axis

The mechanism of tumorigenesis has not been fully identified in laryngeal cancer, which accounts for one fourth of patents with head and neck tumors. Long noncoding RNA PCAT19 has been shown to participate in the prostate cancer progression. However, little is known about the role of PCAT19 in the tumorigenesis of laryngeal cancer. In our study, it was shown that the expression levels of PCAT19 was increased in laryngeal tumor tissues and associated with decreased overall survival. Using laryngeal cancer cells lines HEp-2 and AMC-HN-8, it was demonstrated that knockdown of PCAT19 decreased the cell proliferation, increased the mitochondrial respiration, and inhibited the glycolysis. In detail, it showed that the PDK4 expression and PDHE1α phosphorylation levels were decreased upon the PCAT19 knockdown. Further studies indicated that miR-182 functioned as the bridge between PCAT19 and PDK4, which could also regulate the cellular metabolism thus affecting the cell proliferation. Furthermore, it was shown that the PCAT19/miR-182/PDK4 axis existed and regulated cell proliferation by modulating glycolysis and mitochondrial respiration. Finally, we showed that the PCAT19 knockdown decreased the tumor growth in vivo, possibly through regulating the miR-182/PDK4 axis. In conclusion, we demonstrated that lncRNA PCAT19 promoted cell proliferation and tumorigenesis by modulating the miR-182/PDK4 axis and the metabolism balance. PCAT19 might become a promising new target for laryngeal cancer therapeutics.     

Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.     

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

Ubiquitin-specific protease 4 inhibits mono-ubiquitination of the master growth factor signaling kinase PDK1

Background

Phosphorylation by the phospho-inositide-dependent kinase 1 (PDK1) is essential for many growth factor-activated kinases and thus plays a critical role in various processes such as cell proliferation and metabolism. However, the mechanisms that control PDK1 have not been fully explored and this is of great importance as interfering with PDK1 signaling may be useful to treat diseases, including cancer and diabetes.

Methodology/Principal Findings

In human cells, few mono-ubiquitinated proteins have been described but in all cases this post-translational modification has a key regulatory function. Unexpectedly, we find that PDK1 is mono-ubiquitinated in a variety of human cell lines, indicating that PDK1 ubiquitination is a common and regulated process. Ubiquitination occurs in the kinase domain of PDK1 yet is independent of its kinase activity. By screening a library of ubiquitin proteases, we further identify the Ubiquitin-Specific Protease 4 (USP4) as an enzyme that removes ubiquitin from PDK1 in vivo and in vitro and co-localizes with PDK1 at the plasma membrane when the two proteins are overexpressed, indicating direct deubiquitination.

Conclusions

The regulated mono-ubiquitination of PDK1 provides an unanticipated layer of complexity in this central signaling network and offers potential novel avenues for drug discovery.     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014