Aberrant Activation Of Wnt Signaling Research Articles

Infectious Agents Induce Wnt/β-Catenin Pathway Deregulation in Primary Liver Cancers.

Interaction between infectious agents and liver tissue, as well as repeated and extreme biological events beyond adaptive capacities, may result in pathological conditions predisposing people to development of primary liver cancers (PLCs). In adults, PLCs mainly comprise hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Various infectious agents in the hepatic microenvironment can destabilize normal liver cell functions by modulating the Wnt/β-catenin pathway components. Among them, hepatotropic viruses B, C, and D are involved in Wnt/β-catenin signaling dysregulation. Other microbial agents, including oncogenic viruses such as Epstein-Barr virus (EBV) and human papilloma virus (HPV), bacteria, e.g., Mycoplasma hyorhinis and Salmonella Typhi, the protozoan parasite Toxoplasma gondii, the fungus Aspergillus flavus, and liver flukes such as Clonorchissinensis or Opisthorchis viverrini, may induce malignant transformation in hepatocytes or in target cells of the biliary tract through aberrant Wnt signaling activation. This review focuses on new insights into infectious agents implicated in the deregulation of Wnt signaling and PLC development. Since the Wnt/β-catenin pathway is a driver of cancer following viral and bacterial infections, molecules inhibiting the complex axis of Wnt signaling could represent novel therapeutic approaches in PLC treatment.

A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities.

Aberrant Wnt signaling activation is frequently observed in many cancers. The mutation acquisition of Wnt signaling leads to tumorigenesis, whereas the inhibition of Wnt signaling robustly suppresses tumor development in various in vivo models. Based on the excellent preclinical effect of targeting Wnt signaling, over the past 40 years, numerous Wnt-targeted therapies have been investigated for cancer treatment. However, Wnt signaling-targeting drugs are still not clinically available. A major obstacle to Wnt targeting is the concomitant side effects during treatment due to the pleiotropic role of Wnt signaling in development, tissue homeostasis, and stem cells. Additionally, the complexity of the Wnt signaling cascades across different cancer contexts hinders the development of optimized targeted therapies. Although the therapeutic targeting of Wnt signaling remains challenging, alternative strategies have been continuously developed alongside technological advances. In this review, we give an overview of current Wnt targeting strategies and discuss recent promising trials that have the potential to be clinically realized based on their mechanism of action. Furthermore, we highlight new waves of Wnt targeting that combine recently developed technologies such as PROTAC/molecular glue, antibody-drug conjugates (ADC), and anti-sense oligonucleotides (ASO), which may provide us with new opportunities to target 'undruggable' Wnt signaling.

Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development

Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of β-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.

LSM12 facilitates the progression of colorectal cancer by activating the WNT/CTNNB1 signaling pathway.

Aberrant activation of the WNT signaling pathway is a joint event in colorectal cancer (CRC), but the molecular mechanism is still unclear. Recently, RNA-splicing factor LSM12 (like-Sm protein 12) is highly expressed in CRC tissues. This study aimed to verify whether LSM12 is involved in regulating CRC progression via regulating the WNT signaling pathway. Here, we found that LSM12 is highly expressed in CRC patient-derived tissues and cells. LSM12 is involved in the proliferation, invasion, and apoptosis of CRC cells, similar to the function of WNT signaling in CRC. Furthermore, protein interaction simulation and biochemical experiments proved that LSM12 directly binds to CTNNB1 (also known as β-Catenin) and regulates its protein stability to affect the CTTNB1-LEF1-TCF1 transcriptional complex formation and the associated WNT downstream signaling pathway. LSM12 depletion in CRC cells decreased the in vivo tumor growth through repression of cancer cell growth and acceleration of cancer cell apoptosis. Taken together, we suggest that the high expression of LSM12 is a novel factor leading to aberrant WNT signaling activation, and that strategies targeting this molecular mechanism may contribute to developing a new therapeutic method for CRC.

Expression profile of CD98 heavy chain and L-type amino acid transporter 1 and its prognostic significance in colorectal cancer

L-type amino acid transporter (LAT1) is a neutral amino acid transporter, forming a heterodimer complex with the CD98 heavy chain (CD98hc). In this study, we studied the expression profiles of LAT1 and CD98hc in colorectal cancer (CRC) and its precursor lesions. Transcription levels of CD98hc and LAT1 were significantly increased in CRC compared to the matched normal mucosa. CD98hc and LAT1 expression showed no significant correlations with cancer stem cell markers and intestinal stem cell markers, whereas both had positive correlations with Wnt target genes, AXIN2, and EPHB2, suggesting an association with aberrant Wnt signaling activation. Immunohistochemical analysis revealed that CD98hc and LAT1 are not expressed in normal colonic mucosa and various benign lesions including hyperplastic polyps and sessile and traditional serrated adenomas. CD98hc and LAT1 expressions began to appear in tubular adenomas and further increased in carcinomas. Of interest, CD98hc expression decreased during lymph node metastasis. Survival analysis demonstrated that CD98hc and LAT1 have no significant prognostic effect in CRCs. In conclusion, CD98hc and LAT1 are not normally expressed in colonic mucosa and most benign lesions. Their expression began to appear in tubular adenomas and further increased during the adenoma-to-carcinoma transition. CD98hc expression decreased while metastasizing to regional lymph nodes. However, CD98hc and LAT1 expressions had no prognostic value in patients with CRC.

Epithelial Wnt secretion drives the progression of inflammation-induced colon carcinoma in murine model

SummaryColon cancer is initiated by stem cells that escape the strict control. This process is often driven through aberrant activation of Wnt signaling by mutations in components acting downstream of the receptor complex that unfetter tumor cells from the need for Wnts. Here we describe a class of colon cancer that does not depend on mutated core components of the Wnt pathway. Genetically blocking Wnt secretion from epithelial cells of such tumors results in apoptosis, reduced expression of colon cancer markers, followed by enhanced tumor differentiation. In contrast to the normal colonic epithelium, such tumor cells autosecrete Wnts to maintain their uncontrolled proliferative behavior. In humans, we determined certain cases of colon cancers in which the Wnt pathway is hyperactive, but not through mutations in its core components. Our findings illuminate the path in therapy to find further subtypes of Wnt-dependent colon cancer that might be responsive to Wnt secretion inhibitors.

ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium

Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling.

Autophagy positively regulates Wnt signaling in mice with diabetic retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Aberrant Wnt signaling activation plays a pathological role in DR. However, the underlying mechanisms of aberrant Wnt signaling in DR remain unknown. Autophagy has been reported to be involved in the pathophysiology of DR. The present study aimed therefore to investigate the regulatory effects of autophagy on Wnt signaling in DR. Wnt signaling was activated in the retina of db/db mice combined with an increase in the expression of the autophagic proteins microtubule-associated protein 1A/1B-light chain 3 and beclin-1 and a decrease in the expression of the autophagic protein P62. Inhibition of autophagy by 3-methyladenin decreased Wnt signaling in diabetic retinas, indicating a potential association between Wnt signaling and autophagy. Rapamycin, an autophagy inducer, upregulated Wnt signaling in the retina of normal C57BL/6J mice. In cultured Müller cells, rapamycin induced autophagy and activated Wnt signaling, while chloroquine, an autophagy inhibitor, inhibited autophagy and downregulated Wnt signaling, suggesting that autophagy could regulate Wnt signaling in mice retina and retinal cells. In summary, this study demonstrated that autophagy may positively regulate Wnt signaling in diabetic retinas, indicating a potential mechanism of Wnt signaling upregulation in DR and a possible novel therapeutic target of DR.

Pyrvinium pamoate can overcome artemisinin\u2019s resistance in anaplastic thyroid cancer

BackgroundAnaplastic thyroid carcinoma is a highly lethal subtype of thyroid cancer without effective therapies. Drug resistance in anaplastic thyroid carcinoma poses a significant problem. Although artemisinin exerts antitumor effects, but its efficacy in anaplastic thyroid carcinoma is unknown.MethodsWe used RNA sequencing to identify differentially expressed genes. Next, we determined the cause of ART resistance by testing the expression and activity of β-catenin, and enhanced ART activity with a WNT signaling inhibitor.ResultsArtemisinin suppressed the growth of BHT-101 but not human thyroid anaplastic carcinoma (CAL-62) cells. The mechanism of artemisinin resistance in CAL-62 was associated with the aberrant activation of WNT signaling. Pyrvinium pamoate, an inhibitor of WNT signaling, was used to overcome ART resistance in CAL-62 cells. The combination of artemisinin and pyrvinium pamoate suppressed the growth of CAL-62 cells and induced the apoptosis.ConclusionsOur study is the first to prove the efficacy of ART as monotherapy or in combination with PP in the management of anaplastic thyroid cancer, and that the inhibition of WNT signaling may overcome ART resistance.

Epigenetic Regulation of Wnt Signaling by Carboxamide-Substituted Benzhydryl Amines that Function as Histone Demethylase Inhibitors.

SummaryAberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (β-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.

Chlorquinaldol targets the β-catenin and T-cell factor 4 complex and exerts anti-colorectal cancer activity

Aberrant activation of Wnt signaling plays a critical role in the initiation and progression of colorectal cancer (CRC). Chlorquinaldol (CQD) is a topical antimicrobial agent used to treat skin infections. Little is known about the anticancer activity of CQD and its underlying mechanisms. In this study, CQD was demonstrated to inhibit Wnt/β-catenin signaling through targeting the downstream part of this pathway. The results showed that CQD could inhibit the acetylation of β-catenin and disrupt the interaction of β-catenin with T-cell factor 4 (TCF4), leading to reduced binding of β-catenin to the promoters of Wnt target genes and downregulation of the expression of these target genes. Moreover, treatment with CQD suppressed the proliferation, migration, invasion and stemness of CRC cells. In APCmin/+ mice and CRC cell xenografts, administration of CQD suppressed tumor growth and the expression of Wnt target genes c-Myc and Leucine-rich G protein-coupled receptor-5 (LGR5). These results strongly suggest that CQD may be a promising therapeutic agent in the treatment of CRC.

Androgen receptor with short polyglutamine tract preferably enhances Wnt/β-catenin-mediated prostatic tumorigenesis.

Polyglutamine (polyQ) tract polymorphism within the human androgen receptor (AR) shows population heterogeneity. African American men possess short polyQ tracts significantly more frequently than Caucasian American men. The length of polyQ tracts is inversely correlated with the risk of prostate cancer, age of onset, and aggressiveness at diagnosis. Aberrant activation of Wnt signaling also reveals frequently in advanced prostate cancer, and an enrichment of androgen and Wnt signaling activation has been observed in African American patients. Here, we assessed aberrant expression of AR bearing different polyQ tracts and stabilized β-catenin in prostate tumorigenesis using newly generated mouse models. We observed an early onset oncogenic transformation, accelerated tumor cell growth, and aggressive tumor phenotypes in the compound mice bearing short polyQ tract AR and stabilized β-catenin. RNA sequencing analysis showed a robust enrichment of Myc-regulated downstream genes in tumor samples bearing short polyQ AR versus those with longer polyQ tract AR. Upstream regulator analysis further identified Myc as the top candidate of transcriptional regulators in tumor cells from the above mouse samples with short polyQ tract AR and β-catenin. Chromatin immunoprecipitation analyses revealed increased recruitment of β-catenin and AR on the c-Myc gene regulatory locus in the tumor tissues expressing stabilized β-catenin and shorter polyQ tract AR. These data demonstrate a promotional role of aberrant activation of Wnt/β-catenin in combination with short polyQ AR expression in prostate tumorigenesis and suggest a potential mechanism underlying aggressive prostatic tumor development, which has been frequently observed in African American patients.

Abstract C09: Inhibition of tumor growth and post-treatment regrowth by SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, in combination with standard of care in pancreatic cancer models

Abstract Relapse and treatment resistance rates remain high in pancreatic cancer (PC) with standard-of-care (SOC) chemotherapy regimens, although combining them with targeted drug therapies holds promise for improving treatment outcomes and clinical benefits. The Wnt signaling pathway is implicated in multiple cancer hallmarks, including immune evasion, and regulation and survival of cancer stem cells. Aberrant activation of Wnt signaling is common in PC and many other cancers, making it a therapeutic target of interest. SM08502 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt signaling pathway. These studies were performed to assess the tolerability and efficacy of SM08502 in combination with chemotherapy regimens including gemcitabine (G), paclitaxel (P), and nab-paclitaxel (Nab-P) in xenograft models of PC. First, the effect of oral SM08502 in combination with G or G/P on tumor growth inhibition (TGI) relative to vehicle was assessed over 20-21 days in nude mice (n=6 per group) bearing Capan-1 or HPAFII cell-line-derived xenografts. In Capan-1 xenografts, SM08502 (12.5 mg/kg QD) + G (25 mg/kg Q7D i.p.) induced significant TGI (73%, p=0.009) and was more effective than either treatment alone. In HPAFII xenografts, SM08502 (6.25, 12.5 or 25 mg/kg QD) + G/P (75/30 mg/kg Q7D i.p.) induced ≥ 92% TGI (p<0.001) and tumor regressions in ≥ 4 of 6 mice per group at all tested doses. Next, the effect of initial treatment of SOC (G/Nab-P) alone or combined with SM08502 (6.25, 12.5, or 25 mg/kg QD) on tumor regrowth during an observation phase was assessed in Capan-1 xenografts and a patient-derived xenograft (PDX) model. TGI was calculated both during (treatment phase) and up to 40 days after (observation phase) treatment relative to vehicle and SOC alone, respectively. In Capan-1, SM08502 + G/Nab-P (75/30 mg/kg Q7D) induced strong TGI (88-94%, p<0.001) and increased tumor regressions vs. G/Nab-P at the end of treatment (day 27). In the PDX model, SM08502 + G/Nab-P (50/30 mg/kg Q7D) inhibited tumor growth earlier than G/Nab-P in the treatment phase, but TGI was similar after 21 days of treatment (94-96% p<0.0001). At the end of observation in both the Capan-1 and PDX models, SM08502 + G/Nab-P significantly inhibited tumor regrowth (Capan-1, 25 mg/kg, 73.5%, p=0.003; PDX, 12.5 mg/kg, 71.4%; p=0.01). Additionally, survival was improved with all doses of SM08502 + G/Nab-P in Capan-1 xenografts (p<0.05 vs. G/Nab-P). Based on body weight measurements, all treatments were well tolerated except for SM08502 (25 mg/kg) + G/Nab-P in the PDX model (not carried through observation phase). Oral SM08502 potently inhibited tumor growth in combination with chemotherapy and extended antitumor effects in genetically distinct PC models. These data show that the combined application of SM08502 with SOC therapy has potential to provide clinical benefit in PC. A phase 1 study assessing safety, tolerability, and pharmacokinetics of SM08502 in subjects with advanced solid tumors is ongoing (NCT03355066). Citation Format: Carine Bossard, Igor Astsaturov, Nathalia Cruz, Brian Eastman, Chi-Ching Mak, K.C. Sunil, Betty Tam, Gail Bucci, Josh Stewart, Timothy Phalen, Steven Cha. Inhibition of tumor growth and post-treatment regrowth by SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, in combination with standard of care in pancreatic cancer models [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C09.

Abstract C08: SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates activity against cancer stem cell (CSC)-enriched pancreatic cancer cells and suppresses stemness in vitro

Abstract Cancer stem cells (CSCs) are a rare subpopulation of quiescent tumor cells with the ability to self-renew and form new tumors (stemness) that have been identified in many tumor types, including pancreatic cancer (PC). The presence of CSCs in PC tumors may contribute to chemotherapy resistance and relapse. The Wnt signaling pathway is a highly conserved developmental pathway that has been implicated in the maintenance and survival of CSCs. Aberrant activation of Wnt signaling is common in PC where it may functionally support cell proliferation and tumor-forming capacity; therefore, this pathway is a therapeutic target of interest. SM08502 is a novel, small-molecule pan-CLK inhibitor that has been shown to potently inhibit Wnt pathway activity in preclinical studies. The purpose of these studies was to test the hypothesis that SM08502 could impair CSC viability and stemness in PC cell lines. CSC-enriched tumor spheres were generated by culturing Panc1 cells in nonadherent conditions in which most cells lacking stemness undergo anoikis (programmed cell death). These CSC-enriched Panc1 (Panc1-CSC) cell cultures were analyzed for stemness marker expression via FACS and qPCR to validate the enrichment protocol. The effect of SM08502 on spheroid formation and viability was assessed in comparison to salinomycin and napabucasin as positive controls for CSC inhibition. Inhibition of CSCs within parental PC lines (Capan-1, HPAFII, and Panc1) was assessed by 4-day pretreatment of cells in culture with DMSO or SM08502 and a 4-day recovery period followed by plating 1 or 10 cells/well in 96-well plates under nonadherent conditions and quantifying spheroid formation after 24 days. Anoikis-mediated CSC enrichment increased the percentage of cells expressing the stem cell surface protein marker CXCR4 and increased expression of multiple stemness-related genes. Treatment with either positive control compounds or SM08502 inhibited spheroid formation and decreased the viability of the Panc1-CSC cells. Notably, SM08502 was more potent by EC50 than either salinomycin or napabucasin in the cell viability assay. Expression analyses indicated that SM08502 significantly reduced stemness-related gene expression in Panc1-CSC cultures, including CD24, LGR4, and VDR (p<0.01 vs DMSO). In addition, treatment of Capan-1, HPAFII, and Panc1 cells with SM08502 strongly impaired their ability to form spheres by 8- to 12-fold vs. DMSO, indicating that CSCs within these cell line populations are sensitive to treatment with SM08502. In summary, SM08502 demonstrated anti-CSC activity against PC and appeared more potent than known CSC-inhibiting compounds. By depleting CSCs and reducing stemness in tumors, SM08502 can potentially address relapse and treatment resistance in PC. A phase 1 study assessing safety, tolerability, and pharmacokinetics of SM08502 in subjects with advanced solid tumors is ongoing (NCT03355066). Citation Format: Carine Bossard, Nathalia Cruz, Brian Eastman, Chi-Ching Mak, Sunil KC, Betty Tam, Timothy Phalen, Steven Cha. SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates activity against cancer stem cell (CSC)-enriched pancreatic cancer cells and suppresses stemness in vitro [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C08.

Abstract 3490: Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population

Abstract Background: CRC is the 3rd most commonly diagnosed malignancy and 4th leading cause of cancer related deaths worldwide. Over two decades of extensive research from the West has revealed three major tumorigenesis pathways responsible for CRC viz. a) Chromosomal Instability (CIN) arising from aberrant activation of Wnt signalling, b) Microsatellite Instability (MSI) caused by inactivation of MisMatch Repair (MMR) genes and c) CpG Island Methylator Phenotype resulting in promoter DNA methylation induced inactivation of tumour suppressor genes. Previous results from our laboratory showed a significant proportion of Early Onset Sporadic Rectal Cancer (EOSRC) to be driven neither by aberrant Wnt signalling nor MSI. We also identified a significant proportion of MSI+ familial CRC not exhibiting loss either of the four major MMR genes indicating involvement of addition MMR or non-MMR genes. We therefore endeavoured to determine status of MSI in sporadic CRC samples from the Indian population. We are performing analysis of the status of other molecular features including CIMP, BRAF/KRAS/p53 mutation status, Wnt, etc. to obtain a wholesome molecular profile of sporadic CRC from the Indian population. Methodology: The microsatellite pattern in CRC tumours was determined using the five microsatellite marker panel as per the Bethesda guidelines. Expression status of MMR proteins (MLH1, MSH2, MSH6 & PMS2) was evaluated using immunohistochemistry. Mutations in BRAF (V600E) and KRAS (Codon 12 and 13) were identified by Sanger sequencing. CIMP status is being determined using the MethyLight technology as well as methylation specific PCR. Results: A comprehensive screen of close to 200 CRC samples revealed a significantly higher proportion of sporadic CRC tumors (41%; 50% of colonic and 31% of rectal cancer samples) exhibiting MSI as compared to 14% reported in The Cancer Genome Atlas (TCGA) data. Among MSI-high tumours, only 27% and 15% showed loss of MLH1 or MSH2 respectively, a significantly lower frequency compared to Caucasian data. Instability in the BAT25 microsatellite was significantly high in tumors harboring MLH1 loss. More importantly, a significant proportion of MSI+ CRC tumors were devoid of loss of either of the four major MMR genes. In addition, the BRAF V600E mutation, a known cause of MLH1 silencing, is absent in all but one MSI+ tumors. Work on determining status of CIMP and Wnt signalling is currently underway. Conclusions: Significantly higher proportion of sporadic CRCs from Indian population exhibit MSI but the underlying mechanism appears to be novel and distinct from those identified from the Caucasian population. Deep sequencing based studies are currently underway to determine molecular aberrations driving MSI in the Indian sporadic CRC. Note: This abstract was not presented at the meeting. Citation Format: Ashmala Naz, Vijaya Tourani, Satish Rao, Murali Dharan Bashyam. Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3490.

Alterations in GRHL2-OVOL2-ZEB1 axis and aberrant activation of Wnt signaling lead to altered gene transcription in posterior polymorphous corneal dystrophy

Mutations associated with posterior polymorphous corneal dystrophy (PPCD) have been identified in three genes: ZEB1 (zinc-finger E-box binding homeobox 1) associated with sub-type PPCD3; OVOL2 (ovol-like zinc finger 2) associated with sub-type PPCD1; and GRHL2 (grainyhead like transcription factor 2) associated with sub-type PPCD4. Each of these genes encodes a transcription factor that regulates cell-state transitions. While the discovery of these PPCD-associated genes has greatly expanded our knowledge of the genetic basis of PPCD, the molecular mechanisms via which mutations in these genes lead to indistinguishable disease phenotypes have yet to be elucidated. To characterize the gene expression profiles of the genetic sub-types of PPCD, RNA-seq was performed on corneal endothelium derived from an individual with PPCD1 who harbors a c.-307T > C OVOL2 promoter mutation. Transcriptomic analysis of this and previously-reported RNA-seq data from two individuals with PPCD (the first with PPCD3 associated with a ZEB1 truncating mutation (c.1381delinsGACGAT) and the second with genetically unresolved PPCD in which ZEB1 coding region, OVOL2 promoter and GRHL2 promoter, exon 1, and intron 1 mutations were excluded) revealed: OVOL2 expression increased in PPCD1 (259 fold), unchanged in PPCD3 and slightly increased in genetically unresolved PPCD (from 0 TPM to 0.86 TPM, undefined fold change); ZEB1 expression decreased in PPCD1 (−5.9 fold), PPCD3 (−3.95 fold) and genetically unresolved PPCD (−3.96 fold); and GRHL2 expression increased in PPCD1 (333.5 fold), slightly increased (from 0 TPM to 0.67 TPM, undefined fold change) in PPCD3 and increased in genetically unresolved PPCD (1853 fold). Additionally, as the majority of pedigrees affected with PPCD remain genetically unresolved, we screened the promoter, exon 1, and intron 1 regions of GRHL2 in 24 PPCD probands who do not harbor a ZEB1 or OVOL2 mutation. GRHL2 screening did not identify any novel or rare GRHL2 variant in these 24 individuals. As ZEB1 can act as an activator or repressor of downstream target gene expression depending on Wnt signaling pathway activation or deactivation, we also sought to determine whether or not Wnt signaling is active in PPCD by performing immunohistochemistry in corneal tissue sections derived from an individual affected with PPCD3 and from an individual with genetically unresolved PPCD. Immunohistochemistry results demonstrated corneal endothelial nuclear accumulation of S552 phos-β-catenin and cytosolic localization of S33/37/T42 non-phosphorylated β-catenin in PPCD, indicating aberrant activation of Wnt signaling, which was not observed in control corneal endothelium. These findings suggest that alterations in the ZEB1-OVOL2-GRHL2 axis (caused by PPCD-associated mutations) lead to changes in corneal endothelial cell state and molecular pathways, including the aberrant activation of the Wnt signaling pathway.

Effects of SM08502, a novel, oral small-molecule inhibitor of Wnt pathway signaling, on gene expression and antitumor activity in colorectal cancer (CRC) models.

e15185 Background: Aberrant activation of Wnt signaling contributing to tumorigenesis is most commonly associated with CRC (90% harbor Wnt pathway mutations). SM08502, a novel, oral Wnt signaling pathway inhibitor, was evaluated in preclinical CRC models. Methods: In vitro Wnt signaling: assessed using TOPflash β-catenin/TCF reporter assay in SW480 human CRC cells. In vitro Wnt pathway gene expression: measured by qRT-PCR in SW480 and Wnt3a-stimulated cells (HEK-293T, IEC-6), and with the Nanostring Wnt pathway array (180 genes) across a panel of 16 CRC cell lines. In vitro cell proliferation: 17 CRC cell lines were used to test cell viability following treatment. In vivo antitumor activity: Oral SM08502 was tested in CRC mouse xenografts (SW480, HCT 116) and a PDX model over 20-21 days (QD, QOD). 24-hr pharmacodynamic (PD) analysis of Wnt pathway gene expression was done in SW480 tumor explants from mice following one 25 mg/kg dose. Results: SM08502 inhibited Wnt pathway signaling (EC50 = 46 nM) in SW480 cells. Wnt pathway gene expression was inhibited by SM08502 (0.3-3 µM) in Wnt3a-stimulated cells ( AXIN2, LEF1) and SW480 ( AXIN2, CTNNB1, LEF1, MYC, TCF7, TCF7L2) at 24 hrs ( P < .05 vs. vehicle) . Corresponding effects on protein expression were confirmed for all genes except CTNNB1, suggesting SM08502 acted independently of β-catenin. Nanostring array screening identified inhibition of LRP5, DVL2, BTRC, and ERBB2 by SM08502. Cell proliferation was inhibited in all 17 lines (avg. EC50 = 177 nM). In vivo, SM08502 was well tolerated and induced dose-dependent antitumor effects in xenografts and PDX models. Tumor growth inhibition for 25 mg/kg QD (max dose) was 83%, 56%, and 70% in SW480, HCT 116, and PDX, respectively. PD analysis showed significant inhibition ( P< .05 vs. vehicle) of TCF7, MYC, LRP5, DVL2, and BTRC expression 8 hrs post treatment. Conclusions: In preclinical CRC models, SM08502 was a potent inhibitor of Wnt pathway signaling and gene expression. It showed strong antitumor activity in human tumor models with activating Wnt pathway mutations. The safety, tolerability, and PK of SM08502 are being evaluated in an ongoing phase 1 study (NCT03355066).

Targeting the Wnt/β-catenin pathway in human osteosarcoma cells.

Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

Epigenetic Alterations of Wnt Signaling Pathways in Nasopharyngeal Carcinoma

The purpose of this study is to discuss the epigenetic alterations of Wnt signaling pathways in nasopharyngeal carcinoma. Methylomic analyses have revealed that in nasopharyngeal carcinoma, numerous genes in Wnt signaling pathways are hypermethylated, which results in the inactivation of tumor-suppressor genes and aberrant activation of Wnt signaling. Reactivation of the tumor-suppressor genes by demethylation or ectopic expression inhibits the proliferation, metastasis, or stemness of nasopharyngeal carcinoma cells. Epigenetic alterations of Wnt signaling pathways contribute to cancer-cell stemness, epithelial-mesenchymal transition, and metastasis during the tumorigenesis of nasopharyngeal carcinoma. Methylated DNAs of Wnt signaling factors hold considerable potential as candidate biomarkers for early detection of nasopharyngeal carcinoma.

Luteolin attenuates Wnt signaling via upregulation of FZD6 to suppress prostate cancer stemness revealed by comparative proteomics

The mechanisms underlying luteolin-induced inhibition of prostate cancer (PCa) stemness have remained elusive. Here, we report that luteolin suppresses PCa stemness through Wnt signaling by upregulation of FZD6 (frizzled class receptor 6). Luteolin inhibits PCa cell proliferation, migration, self-renewal as well as the expression of prostate cancer stem cell markers in vitro. Through iTRAQ-based quantitative proteomics study, we identified 208 differentially expressed proteins in luteolin-treated PC-3 cells. Subsequent mechanistic analysis revealed that luteolin inhibits Wnt signaling by transcriptional upregulation of FZD6, and thereby suppressing the stemness of PCa cells. Furthermore, we identified FZD6 as a tumor suppressor that can abolish PCa stemness. In summary, our findings demonstrate that suppression of Wnt signaling by upregulation of FZD6 is a mechanism underlying luteolin-induced inhibition of PCa stemness. Our work suggests a new therapeutic strategy against human prostate cancer caused by aberrant activation of Wnt signaling.