Yong Hoon Cha

MiRNA-34 intrinsically links p53 tumor suppressor and Wnt signaling

Though tumor suppressor p53 and the canonical Wnt cascade have been extensively studied for the last 30 years, due to their important physiological roles, the two signaling pathways have been largely considered independent. Recently, the miR-34 family was found to directly link p53 and Wnt, revealing the tight connection between loss of tumor suppressor function and activation of oncogenic signaling. These observations demonstrate that miR-34, known to be directly downstream of p53, targets a set of highly conserved sites in the UTR of Wnt and EMT genes, specifically WNT1, WNT3, LRP6, AXIN2, β-catenin, LEF1 and Snail, resulting in suppression of TCF/LEF transcriptional activity and the EMT program. The loss of p53 function increases Wnt activities and promotes the Snail-dependent EMT program at multiple levels in a miR-34/UTR-specific manner. The TCF/LEF transcriptional signature was closely associated with functionality of p53 and miR-34 in clinical samples, suggesting the pervasive impact of miR-34 loss on the oncogenic pathway in human cancer. Here, we review recent findings on ceRNA in light of novel data to elucidate the physiological relevance of the p53-miR-34-Wnt network, which encompasses sets of genes and directions of signaling. As loss of wt-p53 or hyperactivation of Wnt is critical in maintaining cancer stem cell properties and in establishing the metastatic program, these observations indicate a mechanism of miR-mediated quasi-sufficiency which connects tumor suppressor and oncogenic signaling pathways, supporting a continuum model of human cancer.

p53 regulates nuclear GSK-3 levels through miR-34-mediated Axin2 suppression in colorectal cancer cells

p53 is a bona fide tumor suppressor gene whose loss of function marks the most common genetic alteration in human malignancy. Although the causal link between loss of p53 function and tumorigenesis has been clearly demonstrated, the mechanistic links by which loss of p53 potentiates oncogenic signaling are not fully understood. Recent evidence indicates that the microRNA-34 (miR-34) family, a transcriptional target of the p53, directly suppresses a set of canonical Wnt genes and Snail, resulting in p53-mediated suppression of Wnt signaling and the EMT process. In this study, we report that p53 regulates GSK-3β nuclear localization via miR-34-mediated suppression of Axin2 in colorectal cancer. Exogenous miR-34a decreases Axin2 UTR-reporter activity through multiple binding sites within the 5′ and 3′ UTR of Axin2. Suppression of Axin2 by p53 or miR-34 increases nuclear GSK-3β abundance and leads to decreased Snail expression in colorectal cancer cells. Conversely, expression of the non-coding UTR of Axin2 causes depletion of endogenous miR-34 via the miR-sponge effect together with increased Axin2 function, supporting that the RNA-RNA interactions with Axin2 transcripts act as an endogenous decoy for miR-34. Further, RNA transcripts of miR-34 target were correlated with Axin2 in clinical data set of colorectal cancer patients. Although the biological relevance of nuclear GSK-3 level has not been fully studied, our results demonstrate that the tumor suppressor p53/miR-34 axis plays a role in regulating nuclear GSK-3 levels and Wnt signaling through the non-coding UTR of Axin2 in colorectal cancer.

Helicobacter pylori CagA promotes Snail-mediated epithelial-mesenchymal transition by reducing GSK-3 activity.

Cytotoxin-associated gene A (CagA) is an oncoprotein and a major virulence factor of H. pylori. CagA is delivered into gastric epithelial cells via a type IV secretion system and causes cellular transformation. The loss of epithelial adhesion that accompanies the epithelial-mesenchymal transition (EMT) is a hallmark of gastric cancer. Although CagA is a causal factor in gastric cancer, the link between CagA and the associated EMT has not been elucidated. Here, we show that CagA induces the EMT by stabilizing Snail, a transcriptional repressor of E-cadherin expression. Mechanistically we show that CagA binds GSK-3 in a manner similar to Axin and causes it to shift to an insoluble fraction, resulting in reduced GSK-3 activity. We also find that the level of Snail protein is increased in H. pylori infected epithelium in clinical samples. These results suggest that H. pylori CagA acts as a pathogenic scaffold protein that induces a Snail-mediated EMT via the depletion of GSK-3.

Catabolic metabolism during cancer EMT.

Aerobic glycolysis is widely accepted as the glucose metabolism for production of biomass such as nucleotides, amino acids, and fatty acids which underlie the anabolic process of cancer cell proliferation. The epithelial–mesenchymal transition (EMT) is a complex cellular mechanism for invasion and metastatic progression in cancer cells. While Snail-mediated EMT regulated by major oncogenic signaling has been well-studied over the last decade, metabolic reprogramming during the EMT has not. In this work, we emphasize the importance of catabolic metabolism for cancer cell survival during cancer cell EMT. Because specific catabolic processes such as autophage and fatty acid oxidation have been well explained, we mainly focus on the general aspects of energy metabolism promoting cancer cell survival under metabolic stress. We also revisit the role of mitochondria in catabolism as oxidative phosphorylation in cancer has long been underestimated. Considering the highly inefficient process of metastatic progression and profound metabolic stress following matrix detachment of solid cancer, catabolic reprogramming during the EMT may play an important role in overcoming metastatic inefficiency of cancer cells.

Snail reprograms glucose metabolism by repressing phosphofructokinase PFKP allowing cancer cell survival under metabolic stress

Dynamic regulation of glucose flux between aerobic glycolysis and the pentose phosphate pathway (PPP) during epithelial–mesenchymal transition (EMT) is not well-understood. Here we show that Snail (SNAI1), a key transcriptional repressor of EMT, regulates glucose flux toward PPP, allowing cancer cell survival under metabolic stress. Mechanistically, Snail regulates glycolytic activity via repression of phosphofructokinase, platelet (PFKP), a major isoform of cancer-specific phosphofructokinase-1 (PFK-1), an enzyme involving the first rate-limiting step of glycolysis. The suppression of PFKP switches the glucose flux towards PPP, generating NADPH with increased metabolites of oxidative PPP. Functionally, dynamic regulation of PFKP significantly potentiates cancer cell survival under metabolic stress and increases metastatic capacities in vivo. Further, knockdown of PFKP rescues metabolic reprogramming and cell death induced by loss of Snail. Thus, the Snail-PFKP axis plays an important role in cancer cell survival via regulation of glucose flux between glycolysis and PPP.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress.

Niclosamide is a potential therapeutic for familial adenomatosis polyposis by disrupting Axin-GSK3 interaction

The epithelial-mesenchymal transition (EMT) is implicated in tumorigenesis and cancer progression, and canonical Wnt signaling tightly controls Snail, a key transcriptional repressor of EMT. While the suppression of canonical Wnt signaling and EMT comprises an attractive therapeutic strategy, molecular targets for small molecules reverting Wnt and EMT have not been widely studied. Meanwhile, the anti-helminthic niclosamide has been identified as a potent inhibitor of many oncogenic signaling pathways although its molecular targets have not yet been clearly identified. In this study, we show that niclosamide directly targets Axin-GSK3 interaction, at least in part, resulting in suppression of Wnt/Snail-mediated EMT. In vitro and in vivo, disruption of Axin-GSK3 complex by niclosamide induces mesenchymal to epithelial reversion at nM concentrations, accompanied with suppression of the tumorigenic potential of colon cancer. Niclosamide treatment successfully attenuates Snail abundance while increasing E-cadherin abundance in xenograft tumor. Notably, oral administration of niclosamide significantly suppressed adenoma formation in an APC-MIN mice model, indicating that niclosamide is an effective therapeutic for familial adenomatosis polyposis (FAP) patients. In this study, we identified a novel target to control the canonical Wnt pathway and Snail-mediated EMT program, and discovered a repositioned therapeutics for FAP patients.

ANO9/TMEM16J promotes tumourigenesis via EGFR and is a novel therapeutic target for pancreatic cancer

Background:Anoctamin (ANO)/transmembrane member 16 (TMEM16) proteins mediate diverse physiological and pathophysiological functions including cancer cell proliferation. The present study aimed to identify the role of ANOs in pancreatic cancer.Methods:In an initial screen of ANOs, ANO9/TMEM16J was overexpressed in pancreatic cancer cells, and its role in the pathogenesis of pancreatic cancer was evaluated using an integrated in vitro and in vivo approach. To determine clinical relevance of the experimental findings, the prognostic value of ANO9 was evaluated in patients with pancreatic cancer.Results:The ANO9 mRNA and protein levels were increased in pancreatic cancer-derived cells. Exogenous expression of ANO9 in PANC-1 cells significantly increased cell proliferation in cell cultures and in mice. In contrast, knockdown of ANO9 in AsPC-1, BxPC-3, and Capan-2 cells strongly inhibited cell proliferation. Mechanistic analysis suggested that physical association of ANO9 with epidermal growth factor receptor (EGFR) underlies ANO9-induced cell proliferation. Knockdown of ANO9 augmented the effects of the EGFR inhibitor and the cytotoxic agent on pancreatic cancer cell proliferation. In addition, high ANO9 expression is a poor prognostic factor in patients with pancreatic cancer.Conclusions:The ANO9/TMEM16J appears to be a clinically useful prognostic marker for pancreatic cancer and a potential therapeutic target.

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner

Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.Hippo and Wnt pathways are important for cancer development, and they can cross talk; however, the mechanisms behind this connection are unknown. Here the authors show that DVL (a scaffold protein in the Wnt pathway) regulates the shuttling of YAP (a key component of the Hippo pathway) between cytoplasm and nucleus in specific tumor suppressor contexts.

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.