PLD1 is a key player in cancer stemness and chemoresistance: Therapeutic targeting of cross-talk between the PI3K/Akt and Wnt/ß-catenin pathways.

The development of chemoresistance is a major challenge in the treatment of several types of cancers in clinical settings. Stemness and chemoresistance are the chief causes of poor clinical outcomes. In this context, we hypothesized that understanding the signaling pathways responsible for chemoresistance in cancers is crucial for the development of novel targeted therapies to overcome drug resistance. Among the aberrantly activated pathways, the PI3K-Akt/Wnt/ß-catenin signaling pathway is clinically implicated in malignancies such as colorectal cancer (CRC) and glioblastoma multiforme (GBM). Aberrant dysregulation of phospholipase D (PLD) has been implicated in several malignancies, and oncogenic activation of this pathway facilitates tumor proliferation, stemness, and chemoresistance. Crosstalk involving the PLD and Wnt/ß-catenin pathways promotes the progression of CRC and GBM and reduces the sensitivity of cancer cells to standard therapies. Notably, both pathways are tightly regulated and connected at multiple levels by upstream and downstream effectors. Thus, gaining deeper insights into the interactions between these pathways would help researchers discover unique therapeutic targets for the management of drug-resistant cancers. Here, we review the molecular mechanisms by which PLD signaling stimulates stemness and chemoresistance in CRC and GBM. Thus, the current review aims to address the importance of PLD as a central player coordinating cross-talk between the PI3K/Akt and Wnt/ß-catenin pathways and proposes the possibility of targeting these pathways to improve cancer therapy and overcome drug resistance.

Enhanced Glutaminolysis Drives Hypoxia-Induced Chemoresistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits severe hypoxia, which is associated with chemoresistance and worse patient outcome. It has been reported that hypoxia induces metabolic reprogramming in cancer cells. However, it is not well known whether metabolic reprogramming contributes to hypoxia. Here, we established that increased glutamine catabolism is a fundamental mechanism inducing hypoxia, and thus chemoresistance, in PDAC cells. An extracellular matrix component-based in vitro three-dimensional cell printing model with patient-derived PDAC cells that recapitulate the hypoxic status in PDAC tumors showed that chemoresistant PDAC cells exhibit markedly enhanced glutamine catabolism compared with chemoresponsive PDAC cells. The augmented glutamine metabolic flux increased the oxygen consumption rate via mitochondrial oxidative phosphorylation (OXPHOS), promoting hypoxia and hypoxia-induced chemoresistance. Targeting glutaminolysis relieved hypoxia and improved chemotherapy efficacy in vitro and in vivo. This work suggests that targeting the glutaminolysis-OXPHOS-hypoxia axis is a novel therapeutic target for treating patients with chemoresistant PDAC. SIGNIFICANCE: Increased glutaminolysis induces hypoxia via oxidative phosphorylation-mediated oxygen consumption and drives chemoresistance in pancreatic cancer, revealing a potential therapeutic strategy of combining glutaminolysis inhibition and chemotherapy to overcome resistance.

Inhibition of phospholipase D1 induces immunogenic cell death and potentiates cancer immunotherapy in colorectal cancer.

Phospholipase D (PLD) is a potential therapeutic target against cancer. However, the contribution of PLD inhibition to the antitumor response remains unknown. We developed a potent and selective PLD1 inhibitor based on computer-aided drug design. The inhibitor enhanced apoptosis in colorectal cancer (CRC) cells but not in normal colonic cells, and in vitro cardiotoxicity was not observed. The inhibitor downregulated the Wnt/ß-catenin signaling pathway and reduced the migration, invasion, and self-renewal capacity of CRC cells. In cancer, therapeutic engagement of immunogenic cell death (ICD) leads to more effective responses by eliciting the antitumor immunity of T cells. The CRC cells treated with the inhibitor showed hallmarks of ICD, including downregulation of "do not eat-me" signals (CD24, CD47, programmed cell death ligand 1 [PD-L1]), upregulation of "eat-me" signal (calreticulin), release of high-mobility group Box 1, and ATP. PLD1 inhibition subsequently enhanced the phagocytosis of cancer cells by macrophages through the surface expression of costimulatory molecules; as a result, the cancer cells were more susceptible to cytotoxic T-cell-mediated killing. Moreover, PLD1 inhibition attenuated colitis-associated CRC and orthotopically injected tumors, probably by controlling multiple pathways, including Wnt signaling, phagocytosis checkpoints, and immune signaling. Furthermore, combination therapy with a PLD1 inhibitor and an anti-PD-L1 antibody further enhanced tumor regression via immune activation in the tumor environment. Collectively, in this study, PLD1 was identified as a critical regulator of the tumor microenvironment in colorectal cancer, suggesting the potential of PLD1 inhibitors for cancer immunotherapy based on ICD and immune activation. PLD1 inhibitors may act as promising immune modulators in antitumor treatment via ICD.

Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

BACKGROUND/AIMS: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. METHODS: A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-ß1 and TGFß type I receptor (TGFßRI), and protein expression was assessed by immunoblotting and immunofluorescence. RESULTS: Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-ß1 expression, and silencing of TGF-ß1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFßRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. CONCLUSION: Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

Self-transducible LRS-UNE-L peptide enhances muscle regeneration.

BACKGROUND: Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C-terminal unique attached sequence motif (UNE) domain of leucyl-tRNA synthetase (LRS-UNE-L) as an mTORC1 (mTOR complex1)-activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle-enhancing peptide. METHODS: In vitro Vps34 lipid kinase assay, phosphatidylinositol 3-phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS-UNE-L on the Vps34-PLD1-mTOR pathway. Adeno-associated virus (AAV)-LRS-UNE-L was transduced in C2C12 cells in vitro, in BaCl2 -injured tibialis anterior (TA) muscles, and in 18-month-old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle-specific cell-permeable peptide M12 was fused with LRS-UNE-L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12-LRS-UNE-L was introduced into BaCl2 -injured TA muscles of 15-week-old Pld1+/+ or Pld1-/- mice, and its effect was analysed by measurement of cross-sectional area of regenerating muscle fibres. RESULTS: The LRS-UNE-L expression restored amino acid-induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases-independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS-UNE-L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid-treated control cells, but it did not affect autophagic flux. AAV-delivered LRS-UNE-L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin-like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV-LRS-UNE-L increased the size of regenerating muscle fibres in BaCl2 -injured TA muscles (124%, P = 0.0279) (n = 9-10), but it did not change the muscle fibre size of TA muscles in old mice. M12-LRS-UNE-L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2 -injured TA muscles in a PLD1-dependent manner (116%, P = 0.0022) (n = 6). CONCLUSIONS: Our results provide compelling evidence that M12-LRS-UNE-L could be a muscle-enhancing protein targeting mTOR.

Phospholipase D2 is a positive regulator of sirtuin 1 and modulates p53-mediated apoptosis via sirtuin 1.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent histone deacetylase that plays diverse physiological roles. However, little is known about the regulation of SIRT1 activity. Here, we show that phospholipase D2 (PLD2), but not PLD1, selectively interacts with SIRT1 and increases the deacetylase activity of SIRT1. PLD2 does not interact with the other isozymes of SIRT (SIRT2-7). Two leucine residues in the LXXLL motif (L173 and L174) in the phox domain of PLD2 interact with the region essential for SIRT1 activity. PLD2 stimulates the SIRT1-mediated deacetylation of p53 independent of its lipase activity. In our study, mutagenesis of the LXXLL motif suppressed the interaction of PLD2 with SIRT1 and inhibited SIRT1-mediated p53 deacetylation and p53-induced transactivation of proapoptotic genes. Ultimately, overexpression of wild-type PLD2 but not that of LXXLL-mutant PLD2 protected cells against etoposide-induced apoptosis. Moreover, PLD2 did not protect against apoptosis induced by SIRT1 depletion under genotoxic stress. Collectively, our results suggest that PLD2 is a positive regulator of SIRT1 and modulates p53-mediated apoptosis via SIRT1.

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, ß-catenin-osteoprotegerin, PPAR-γ and C/EBPα signaling axis.

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1/- MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1-/- bone marrow-derived macrophages. Pld1-/- osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1-/- mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1-/- osteoblasts reduced ß-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.

Phospholipase D1 is upregulated by vorinostat and confers resistance to vorinostat in glioblastoma.

Glioblastoma (GBM) is an aggressive brain tumor and drug resistance remains a major barrier for therapeutics. Epigenetic alterations are implicated in GBM pathogenesis, and epigenetic modulators including histone deacetylase (HDAC) inhibitors are exploited as promising anticancer therapies. Here, we demonstrate that phospholipase D1 (PLD1) is a transcriptional target of HDAC inhibitors and confers resistance to HDAC inhibitor in GBM. Treatment of vorinostat upregulates PLD1 through PKCζ-Sp1 axis. Vorinostat induces dynamic changes in the chromatin structure and transcriptional machinery associated with PLD1 promoter region. Cotreatment of vorinostat with PLD1 inhibitor further attenuates invasion, angiogenesis, colony-forming capacity, and self-renewal capacity, compared with those of either treatment. PLD1 inhibitor overcomes resistance to vorinostat in GBM cells intracranial GBM tumors. Our finding provides new insight into the role of PLD1 as a target of resistance to vorinostat, and PLD1 inhibitor might provide the basis for therapeutic combinations with improved efficacy of HDAC inhibitor.

Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase.

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK. [BMB Reports 2021; 54(2): 112-117].

Thermosensitive Hydrogel Harboring CD146/IGF-1 Nanoparticles for Skeletal-Muscle Regeneration.

In skeletal-muscle regeneration, it is critical to promote efferocytosis of immune cells and differentiation of satellite cells/postnatal muscle stem cells at the damaged sites. With the optimized poloxamer 407 composition gelled at body temperature, the drugs can be delivered locally. The purpose of this study is to develop a topical injection therapeutic agent for muscle regeneration, sarcopenia, and cachexia. Herein, we construct an injectable, in situ hydrogel system consisting of CD146, IGF-1, collagen I/III, and poloxamer 407, termed CIC gel. The secreted CD146 then binds to VEGFR2 on the muscle surface and effectively induces efferocytosis of neutrophils and macrophages. IGF-1 promotes satellite cell differentiation, and biocompatible collagen evades immune responses of the CIC gel. Consequently, these combined molecules activate muscle regeneration via autophagy and suppress muscle inflammation and apoptosis. Conclusively, we provide an applicable concept of the myogenesis-activating protein formulation, broadening the thermoreversible hydrogel to protein therapeutics for damaged muscle recovery.

PLD1 and PLD2 differentially regulate the balance of macrophage polarization in inflammation and tissue injury.

Phospholipase D (PLD) isoforms PLD1 and PLD2 serve as the primary nodes where diverse signaling pathways converge. However, their isoform-specific functions remain unclear. We showed that PLD1 and PLD2 selectively couple to toll-like receptor 4 (TLR4) and interleukin 4 receptor (IL-4R) and differentially regulate macrophage polarization of M1 and M2 via the LPS-MyD88 axis and the IL-4-JAK3 signaling, respectively. Lipopolysaccharide (LPS) enhanced TLR4 or MyD88 interaction with PLD1; IL-4 induced IL-4R or JAK3 association with PLD2, indicating isozyme-specific signaling events. PLD1 and PLD2 are indispensable for M1 polarization and M2 polarization, respectively. Genetic and pharmacological targeting of PLD1 conferred protection against LPS-induced sepsis, cardiotoxin-induced muscle injury, and skin injury by promoting the shift toward M2; PLD2 ablation intensified disease severity by promoting the shift toward M1. Enhanced Foxp3+ regulatory T cell recruitment also influenced the anti-inflammatory phenotype of Pld1LyzCre macrophages. We reveal a previously uncharacterized role of PLD isoforms in macrophage polarization, signifying potential pharmacological interventions for macrophage modulation.

Nitration of protein phosphatase 2A increases via Epac1/PLCε/CaMKII/HDAC5/iNOS cascade in human endometrial stromal cell decidualization.

Decidualization of the endometrial stroma is an essential differentiation process for embryo implantation and maintenance of pregnancy. We previously reported that protein phosphatase 2A (PP2A) acts as a key mediator during cAMP-induced decidualization of human endometrial stromal cells (hESCs). However, the mechanism underlying its activation has remained obscure in hESCs. In the present study, we aimed to reveal the mechanism that induces the nitration of PP2A catalytic subunit (PP2Ac) during cAMP-induced decidualization of hESCs. First, cAMP-induced PP2Ac nitration was significantly repressed using L-NAME, an inhibitor of nitric oxide synthase (NOS). Among several NOS isoforms, only inducible NOS (iNOS) was highly expressed in hESCs, indicating that iNOS directly induces the nitration of PP2Ac. Second, cAMP-induced iNOS expression and PP2Ac nitration were decreased by treatment with TSA, an inhibitor of histone deacetylase 5 (HDAC5). cAMP-induced phosphorylation of CaMKII and HDAC5 was suppressed by treatment with U73122 (an inhibitor of phospholipase C) or transfection of PLCε siRNA. Finally, small G protein Rap1 and its guanine nucleotide exchange factor Epac1 were found to be involved in cAMP-induced PP2A activation. Taken together, our results suggest that PP2Ac nitration during cAMP-induced decidualization of hESCs is induced through the Epac1-Rap1-PLCε-CaMKII-HDAC5-iNOS signaling pathway.

Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear. METHODS: In this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model. RESULTS: HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment. CONCLUSION: These findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

Upregulation of DR5 and Downregulation of Survivin by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated Apoptosis in Renal Cancer Cells via Ubiquitin-Proteasome Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Phospholipase D1 inhibition sensitizes glioblastoma to temozolomide and suppresses its tumorigenicity.

Resistance of glioblastoma to the chemotherapeutic compound temozolomide is associated with the presence of glioblastoma stem cells in glioblastoma and is a key obstacle for the poor prognosis of glioblastoma. Here, we show that phospholipase D1 is elevated in CD44High glioblastoma stem cells and in glioblastoma, especially recurring glioblastoma. Phospholipase D1 elevation positively correlated with the level of CD44 and poor prognosis in glioblastoma patients. Temozolomide significantly upregulated the expression of phospholipase D1 in the low and moderate CD44 populations of glioblastoma stem cells, but not in the CD44High population in which phospholipase D1 is highly expressed. Phospholipase D1 conferred resistance to temozolomide in CD44High glioblastoma stem cells and increased their self-renewal capacity and maintenance. Phospholipase D1 expression significantly correlated with levels of temozolomide resistance factors, which were suppressed by microRNA-320a and -4496 induced by phospholipase D1 inhibition. Genetic and pharmacological targeting of phospholipase D1 attenuated glioblastoma stem cell-derived intracranial tumors of glioblastoma using the microRNAs, and improved survival. Treatment solely with temozolomide produced no benefits on the glioblastoma, whereas in combination, phospholipase D1 inhibition sensitized glioblastoma stem cells to temozolomide and reduced glioblastoma tumorigenesis. Together, these findings indicate that phospholipase D1 inhibition might overcome resistance to temozolomide and represents a potential treatment strategy for glioblastoma. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer.

BACKGROUND: Stabilization of RAS is a key event for the hyper-activation of Wnt/ß-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. METHODS: We generated mice with deletion of Wdr76 (Wdr76-/-) and crosses of Wdr76-/- with ApcMin/+ (Wdr76-/-; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture. RESULTS: Wdr76-/- mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76-/-; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and ß-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival. CONCLUSIONS: In analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.

A Therapeutic Strategy for Chemotherapy-Resistant Gastric Cancer via Destabilization of Both ß-Catenin and RAS.

: Treatment of advanced gastric cancer patients with current standard chemotherapeutic agents frequently results in resistance, leading to poor overall survival. However, there has been no success in developing strategies to overcome it. We showed the expression levels of both ß-catenin and RAS were significantly increased and correlated in tissues of 756 gastric cancer (GC) patients and tissues of primary- and acquired-resistance patient-derived xenograft tumors treated with 5-fluorouracil and oxaliplatin modulated with leucovorin (FOLFOX). On the basis of our previous studies, where small molecules to suppress colorectal cancer (CRC) via degrading both ß-catenin and RAS were developed, we tested the effectiveness of KYA1797K, a representative compound functioning by binding axin, in the growth of GC cells. The efficacy test of the drugs using gastric tumor organoids of Apc1638N mice showed that the CD44 and ALDH1A3 cancer stem cell markers were induced by FOLFOX, but not by KYA1797K. KYA1797K also efficiently suppressed tumors generated by re-engrafting the FOLFOX-resistant patient-derived xenograft (PDX) tumors, which also showed resistance to paclitaxel. Overall, the small-molecule approach degrading both ß-catenin and RAS has potential as a therapeutic strategy for treating GC patients resistant to current standard chemotherapies.

A Ras destabilizer KYA1797K overcomes the resistance of EGFR tyrosine kinase inhibitor in KRAS-mutated non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are widely used for treatment of non-small cell lung cancer (NSCLC), but they have shown limited efficacy in an unselected population of patients. The KRAS mutations, which are identified in approximately 20% of NSCLC patients, have shown to be associated with the resistance to the EGFR tyrosine kinase inhibitors (TKIs). Currently, there is no clinically available targeted therapy which can effectively inhibit NSCLC tumors harboring KRAS mutations. This study aims to show the effectiveness of KYA1797K, a small molecule which revealed anti-cancer effect in colorectal cancer by destabilizing Ras via inhibiting the Wnt/ß-catenin pathway, for the treatment of KRAS-mutated NSCLC. While erlotinib fail to have anti-transforming effect in NSCLC cell lines harboring KRAS mutations, KYA1797K effectively inhibited the Ras-ERK pathway in KRAS-mutant NSCLC cell lines. As a result, KYA1797K treatment suppressed the growth and transformation of KRAS mutant NSCLC cells and also induced apoptosis. Furthermore, KYA1797K effectively inhibited Kras-driven tumorigenesis in the KrasLA2 mouse model by suppressing the Ras-ERK pathway. The destabilization of Ras via inhibition of the Wnt/ß-catenin pathway is a potential therapeutic strategy for KRAS-mutated NSCLC that is resistant to EGFR TKI.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation.

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76-/- mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

ß-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3ß.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.