Ultrasound in Obstetrics & GynecologyAccepted Articles Letter to the Editor Ultrasound prediction of fetal sacrococcygeal teratoma perinatal morbidity J. L. Munoz, Corresponding Author J. L. Munoz [email protected] orcid.org/0000-0003-0431-5280 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USACorrespondence to: Dr J. L. Munoz, Texas Children's Hospital, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, 6621 Fannin Street, Houston, TX 770, (e-mail: [email protected])Search for more papers by this authorC. Buskmiller, C. Buskmiller Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorA. A. Nassr, A. A. Nassr orcid.org/0000-0002-1924-7965 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorM. Sanz Cortes, M. Sanz Cortes orcid.org/0000-0002-0265-9859 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorS. Keswani, S. Keswani Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorA. King, A. King Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorT. Lee, T. Lee Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorM. A. Belfort, M. A. Belfort orcid.org/0000-0001-7887-5737 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorL. Joyeux, L. Joyeux Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorR. V. Donepudi, R. V. Donepudi Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this author J. L. Munoz, Corresponding Author J. L. Munoz [email protected] orcid.org/0000-0003-0431-5280 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USACorrespondence to: Dr J. L. Munoz, Texas Children's Hospital, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, 6621 Fannin Street, Houston, TX 770, (e-mail: [email protected])Search for more papers by this authorC. Buskmiller, C. Buskmiller Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorA. A. Nassr, A. A. Nassr orcid.org/0000-0002-1924-7965 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorM. Sanz Cortes, M. Sanz Cortes orcid.org/0000-0002-0265-9859 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorS. Keswani, S. Keswani Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorA. King, A. King Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorT. Lee, T. Lee Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorM. A. Belfort, M. A. Belfort orcid.org/0000-0001-7887-5737 Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorL. Joyeux, L. Joyeux Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this authorR. V. Donepudi, R. V. Donepudi Department of Obstetrics and Gynecology, Division of Fetal Therapy and Surgery, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA Texas Children's Fetal Center, Department of Obstetrics and Gynecology, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USASearch for more papers by this author First published: 16 February 2024 https://doi.org/10.1002/uog.27617 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/uog.27617. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Supporting Information Filename Description uog27617-sup-0001-TableS1.docxWord 2007 document , 13.2 KB Table S1 Multivariate analysis Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Accepted ArticlesAccepted, unedited articles published online and citable. 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Oral communication abstracts 33.3-39.9)weeks and a median surgical time of 24.5 (12-35) min.In 4 cases, airway patency was confirmed during laryngoscopy and babies were immediately delivered.In 8/12 cases (66.7%), airway obstruction was confirmed during fetoscopy and FETI was successfully performed at a median GA of 36.4 (33.3-38.7)weeks, with a median surgery time of 25.0 (range, 12-45) min.No case required an EXIT procedure.All patients underwent conventional Caesarean delivery with no maternal complications and all neonates were admitted to neonatal intensive care unit with a correctly positioned endotracheal tube (ETT) immediately after delivery.Three neonatal deaths (37.5%) were reported due to complications related to postnatal unplanned extubation, ETT replacement, and neonatal tumoral bleeding respectively.Conclusions: In fetuses with cervical masses and suspected airway obstruction, management with fetal laryngoscopy followed by FETI is feasible and could replace EXIT procedures with good maternal and perinatal outcomes.OC07.07

Systemic therapy is the current standard of care for unresectable advanced hepatocellular carcinoma (HCC), the most malignant form of liver cancer. Clinical practice guideline for HCC includes the management of the second-line immunotherapies after patients become unresponsive to first-line targeted therapies using tyrosine kinase inhibitors (TKI), which is expected to generate optimal therapeutic effect due to the distinct mechanism of action between TKIs and immunotherapies. However, evidence from clinical trials suggests that the response to immunotherapy following TKI treatment is often compromised in TKI-resistant HCC as compared with treatment-naïve HCC, indicating a potential cross-resistance to immunotherapy, which impedes the optimal therapeutic effect of the second-line immunotherapies. In the present study, we first explored the therapeutic effect of anti-PD-1 immunotherapy in TKI-resistant HCC and successfully validated the putative cross-resistance to immunotherapy after TKI treatment. We further observed that TKI-resistant HCC exhibits an immune-evasive microenvironment, characterized by decreased CD8+ T cell and dendritic cell infiltration. This is potentially caused by the intratumoral activation of receptor tyrosine kinase AXL and its downstream target PDPK1 which protect TKI-resistant HCC cells from mitochondrial oxidative stress and the cytosolic leakage of mitochondrial DNA, which subsequently suppresses cGAS-STING together with its downstream interferon pathways and immunogenic cell death. Genetic manipulation of AXL and PDPK1 in TKI-resistant HCC cells abrogated the protective effect and thus activated the immunogenic responses. Blockade of AXL with a selective inhibitor BGB324 re-sensitized TKI-resistant HCC to anti-PD-1 treatment. The current findings suggest that targeting AXL/PDPK1 signaling axis might overcome cross-resistance to second-line immunotherapy for HCC patients who progress on TKI treatment. Citation Format: Yunong Xie, Bosheng Huang, Huajian Yu, Jia-Jian Loh, Rainbow Wing-Hei Leung, Terence Lee, Stephanie Ma, Man Tong. Resistance to tyrosine kinase inhibitor confers an immunosuppressive microenvironment and cross-resistance to immunotherapy through AXL/PDPK1 axis in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 395.

Supplementary figure 1. BQ323636.1-overexpresion stable cell lines were produced using lentiviral system; Supplementary figure 2. BQ323636.1 overexpression conferred resistance to tamoxifen in vivo using xenografts established with MCF-7; Supplementary figure 3. BQ323636.1 overexpressed did not significantly affect the expressions of NCOR2, GPS2, TBLR1 nor HDAC3; Supplementary figure 4. Chromatin Immunoprecipitation (ChIP) assays show overexpression of BQ323636.1 could reduce the amount of NCOR2 interacting with estrogen responsive element of four genes; Supplementary figure 5. The clinical significance of nuclear expression level of BQ323636.1 in the Hong Kong cohort.; Supplementary figure 6. The clinical significance of nuclear expression level of BQ323636.1 in the United Kingdom cohort; Supplementary figure 7. The significance of nuclear expression level of BQ323636.1 on disease free survival.; Supplementary Table 1. Clinical characteristics of patients with Tamoxifen response information available, N= 358

AbstractHyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are members of the voltage‐gated cation channel family known to be expressed in the heart and central nervous system. Ivabradine, a small molecule HCN channel‐blocker, is FDA‐approved for clinical use as a heart rate‐reducing agent. We found that HCN2 and HCN3 are overexpressed in breast cancer cells compared with normal breast epithelia, and the high expression of HCN2 and HCN3 is associated with poorer survival in breast cancer patients. Inhibition of HCN by Ivabradine or by RNAi, aborted breast cancer cell proliferation in vitro and suppressed tumour growth in patient‐derived tumour xenograft models established from triple‐negative breast cancer (TNBC) tissues, with no evident side‐effects on the mice. Transcriptome‐wide analysis showed enrichment for cholesterol metabolism and biosynthesis as well as lipid metabolism pathways associated with ER‐stress following Ivabradine treatment. Mechanistic studies confirmed that HCN inhibition leads to ER‐stress, in part due to disturbed Ca2+ homeostasis, which subsequently triggered the apoptosis cascade. More importantly, we investigated the synergistic effect of Ivabradine and paclitaxel on TNBC and confirmed that both drugs acted synergistically in vitro through ER‐stress to amplify signals for caspase activation. Combination therapy could suppress tumour growth of xenografts at much lower doses for both drugs. In summary, our study identified a new molecular target with potential for being developed into targeted therapy, providing scientific grounds for initiating clinical trials for a new treatment regimen of combining HCN inhibition with chemotherapy.

Abstract Hepatocellular carcinoma (HCC) is the most malignant form of primary liver cancers with poor prognosis. Majority of the cases are diagnosed in advanced stages where therapeutic options are limited and the response rates are low. There is an unmet clinical need for more effective therapeutic treatment. AXL is a receptor tyrosine kinase which belongs to the family of TAM receptors. Overexpression of AXL has been reported in multiple cancers, including HCC. We and others have also reported an up-regulation of AXL in sorafenib-resistant HCC. However, its role in regulating anti-tumor immunity in HCC is not fully understood. In this study, we observed an immunosuppressive gene signature in sorafenib-resistant HCC by RNA-Seq and pathway enrichment analysis. Genetic manipulation and pharmacological inhibition of AXL promoted immunogenic cell death in sorafenib-resistant HCC cells, as evident by increased surface calreticulin and HMGB1 secretion. PDK1, a key modulatory kinase downstream of AXL in the PI3K/Akt pathway, was also up-regulated in sorafenib-resistant HCC. Knockdown of PDK1 similarly led to an increased immunogenic cell death. In a sorafenib-resistant immunocompetent mouse HCC model, blockade of AXL with a selective AXL inhibitor increased tumor-infiltrating activated and proliferating CD8+ T cells, resulting in tumor suppression and prolonged survival of the mice. Taken together, our findings suggest that targeting the AXL/PDK1 signaling axis could be a viable therapeutic option which enhances anti-tumor immunity for the treatment of HCC. Citation Format: Yunong Xie, Lei Zhou, Cheuk Yin Lin, Terence Lee, Jin Ding, Stephanie Ma, Man Tong. Targeting AXL/PDK1 signaling axis activates immunogenic cell death in liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P236.

Background & Aims: Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2 and elevated RSK2 expression has been reported to support oncogenic functions in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC. Methods: We performed exome sequencing and targeted DNA sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential down-stream pathways underlying RSK2 mutations were investigated by RNA sequencing, qRT-PCR and mass spectrometry. Results: We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts and revealed that, among many cancer types, HCC was the cancer most commonly harboring RSK2 mutations. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring RSK2 expression in natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2-inactivating mutations attenuated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced cholesterol biosynthesis-related gene expression in HCC cells. Conclusions: Our findings reveal the mechanistic and functional significance of RSK2-inactivating mutations in HCC. These inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC. Lay summary: In this study, we identified and functionally characterized RSK2-inactivating mutations in human hepatocellular carcinoma and demonstrated their association with aggressive tumor behavior. Mutations in RSK2 drive signaling pathways with known oncogenic potential, leading to enhanced cholesterol biosynthesis and potentially sensitizing tumors to sorafenib treatment. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Compare perinatal and neurological outcomes between a fetoscopic myelomeningocele single-layer closure (SLC) and a three-layer closure (3LC) technique. An exteriorised uterus fetoscopic SLC technique was the initial approach used in our centre, but due to a high rate of CSF leak at birth, a 3LC was developed. Retrospective case control study: 32 consecutive SLC controls were compared with 18 consecutive 3LC cases performed in the same centre. All patients satisfied MOMS trial surgical eligibility criteria and had similar clinical management protocols. All cases had an exteriorised uterus approach, using 2/3 ports and CO2 for uterine insufflation. SLC consisted of a unified skin and dural closure in a single layer; the 3LC comprised 3 distinct layers: dural patch, myofascial layer and skin closure. Perinatal outcomes were similar between both groups. SLC showed a higher rate of CSF leak (8/32 (25%) vs. 3LC (0/17 (0%);p = 0.02), lower rate of reversal of hind brain herniation (18/30 (60%) vs 14/15 (93%);p = 0.02), and higher rate of hydrocephalus based on MOMS trial criteria (23/31 (74%) vs 3/11 (27%);p < 0.01). In their first year of life 15/32 (47%) SLC vs. 3/13 (23%) 3LC cases were treated for hydrocephalus (p = 0.14). Compared to SLC, 3LC preserves the fetal and obstetric benefits of fetoscopic repair, and shows improved neonatal neurological outcomes. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently down-regulated in hepatocellular carcinoma (HCC) cells and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis and therapy resistance potential of HCC cells. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a DEN+CCL4 HCC induced PRMT6-/- mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and that PRMT6 interacted with CRAF, and likely other RAF family members, and their methylation at conserved arginine 100, negatively regulating its activity, and as a consequence resulting in enhanced MEK/ERK signaling. Our work uncovered a critical repressive function for PRMT6 in maintenance of HCC cells by regulating the MEK/ERK pathway via arginine methylation of RAF, providing a new avenue of molecular mechanism by which ERK mediated stemness in HCC cells are developed. Citation Format: LH Chan, L Zhou, Kai Yu Ng, TL Wong, TK Lee, YP Ching, YF Yuan, D Xie, S Richard, MS Huen, XY Guan, S Ma. Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4479.

AbstractPurpose: Adjuvant tamoxifen treatment revolutionized the management of estrogen receptor (ER)–positive breast cancers to prevent cancer recurrence; however, drug resistance compromises its clinical efficacy. The mechanisms underlying tamoxifen resistance are not fully understood, and no robust biomarker is available to reliably predict those who will be resistant. Here, we study BQ323636.1, a novel splice variant of the NCOR2 gene, and evaluate its efficacy in predicting tamoxifen resistance in patients with breast cancer.Experimental Design: A monoclonal anti-BQ323636.1 antibody that specifically recognizes the unique epitope of this splice variant was generated for in vitro mechanistic studies and for in vivo analysis by immunohistochemistry on tissue microarrays of two independent cohorts of 358 patients with more than 10 years clinical follow-up data, who had ER-positive primary breast cancer and received adjuvant tamoxifen treatment. An orthotopic mouse model was also used.Results: Overexpression of BQ323636.1 conferred resistance to tamoxifen in both in vitro and in an orthotopic mouse model. Mechanistically, coimmunoprecipitation showed BQ323636.1 could bind to NCOR2 and inhibit the formation of corepressor complex for the suppression of ER signaling. Nuclear BQ3232636.1 overexpression in patients samples was significantly associated with tamoxifen resistance (P = 1.79 × 10−6, sensitivity 52.9%, specificity 72.0%). In tamoxifen-treated patients, nuclear BQ323636.1 overexpression was significantly correlated with cancer metastasis and disease relapse. Nuclear BQ323636.1 was also significantly associated with poorer overall survival (P = 1.13 × 10−4) and disease-specific survival (P = 4.02 × 10−5).Conclusions: These findings demonstrate that BQ323636.1 can be a reliable biomarker to predict tamoxifen resistance in patients with ER-positive breast cancer. Clin Cancer Res; 24(15); 3681–91. ©2018 AACR.See related commentary by Jordan, p. 3480

Identification and characterization of functional molecular targets conferring stemness properties in hepatocellular carcinoma (HCC) offers crucial insights to overcome the major hurdles of tumor recurrence, metastasis and chemoresistance in clinical management. In the current study, we investigated the significance of Cripto-1 in contributing to HCC stemness. Cripto-1 was upregulated in the sorafenib-resistant clones derived from HCC cell lines and patient-derived xenograft that we previously developed, suggesting an association between Cripto-1 and stemness. By in vitro experiments, Cripto-1 fostered cell proliferation, migration, and invasion. It also enhanced self-renewal ability and conferred chemoresistance of HCC cells. Consistently, silencing of Cripto-1 suppressed in vivo tumorigenicity on serial transplantation. On the downstream signaling mechanism, expression of major components of Wnt/β-catenin pathway β-catenin, AXIN2, and C-MYC, accompanied by β-catenin activity was reduced upon Cripto-1 knockdown. The suppressive effects on stemness properties with Cripto-1 knockdown in vitro and in vivo were partially rescued by forced expression of constitutively active β-catenin. Further elucidation revealed the binding of Cripto-1 to Frizzled-7 (FZD7), low-density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled-3 (DVL3) of the Wnt/β-catenin pathway and stabilized DVL3 protein. Analyses with clinical samples validated Cripto-1 overexpression in HCC tissues, as well as a positive correlation between Cripto-1 and AXIN2 expressions. High Cripto-1 level in tumor was associated with poorer disease-free survival of HCC patients. Taken together, Cripto-1 binds to FZD7/LRP6 and DVL3, stabilizes DVL3 expression and activates the Wnt/β-catenin signaling cascade to confer stemness in HCC. Our study findings substantiated the role of Cripto-1 in determining stemness phenotypes of HCC and mechanistically in modulating the Wnt/β-catenin signaling cascade, one of the most frequently deregulated pathways in liver cancer.

Background: Hepatocellular carcinoma (HCC) is a biologically aggressive cancer.Limitations of current treatment modalities necessitate the elucidation of its pathogenesis at a molecular level.Accumulating evidence suggest that stem cell-like phenotypes are largely responsible for the high recurrence, frequent metastasis and chemoresistance in HCC.Thus identification of functional molecular targets for HCC stemness is a promising direction to devise targeted therapy.Our group previously developed sorafenib-resistant HCC cells in vitro and in vivo by enriching liver tumor-initiating cells through continuous exposure to sorafenib (Lo J, et al.Hepatology 2015;62:534-45).Cripto-1 transcript level was consistently upregulated in the sorafenib-resistant clones derived from HCC cell lines and patient-derived xenograft.In this study we proceeded to characterize the role of Cripto-1 in hepatocarcinogenesis. Methods: Expression of Cripto-1 in 77 clinical HCC samples was assessed by real-time quantitative PCR.Stable knockdown clones were established in HCC cell lines Huh7 and PLC/PRF/5 by a lentiviral-based approach.Cell proliferation, migration and invasion transwell assays were employed to study the functional effects.Self-renewal ability and chemosensitivity were examined by non-adherent sphere formation assay and apoptosis assay using flow cytometry respectively.In vivo tumorigenicity was investigated by a subcutaneous inoculation model with NOD/SCID mice.Activity of Wnt/β-catenin pathway was measured by TOP/ FOPFlash reporter assay.Interaction between Cripto-1 and Dishevelled-3 (DVL3) was tested by co-immunoprecipitation experiment.Results: Cripto-1 was upregulated in 57% cases from our HCC clinical cohort.Silencing of Cripto-1 in HCC cells downregulated stemness markers and chemoresistance-associated genes.Functionally, knockdown of Cripto-1 suppressed cell proliferation, migration, invasion, tumorsphere formation, and sensitized HCC cells to cisplatin and sorafenib.Consistently, silencing of Cripto-1 suppressed in vivo tumorigenicity in terms of tumor incidence, tumor mass and latency period in a limited dilution manner.On elucidating the molecular mechanism we found that expression of major components of Wnt/β-catenin pathway DVL3, β-catenin, Axin2 and c-myc, together with β-catenin activity, was reduced upon Cripto-1 knockdown.The suppressive effects on self-renewal and tumorigenicity were partially rescued by forced expression of constitutively active β-catenin in Cripto-1 knockdown cells.Furthermore, we identified DVL3 as a binding

Dishevelled-3 (Dvl3) is regarded as a binding hub with many different interacting partners. However, its regulation and mechanism on cancer stemness remain to be explored. In this study, we showed that Dvl3 was significantly overexpressed in human hepatocellular carcinomas (HCCs) and promoted cancer stemness both in vitro and in vivo. We found that the non-phosphorylated (NP)-Dvl3 was more stable than the phosphorylated form, more active in activating β-catenin transcriptional activity, and more potent in enhancing self-renewal ability in HCC cells. Mechanistically, we confirmed that the homeodomain-interacting protein kinase-2 (HIPK2) and E3 ubiquitin ligase ITCH were able to physically bind to Dvl3 protein. Knockdown of HIPK2 and the protein phosphatase regulatory unit C-alpha (PP1Cα) resulted in sustained Dvl3 phosphorylation and hence decrease in the NP form of Dvl3. On the other hand, knockdown of E3 ubiquitin ligase ITCH reduced the phosphorylation-induced degradation and stabilized the phosphorylated Dvl3 protein. Furthermore, the NP-Dvl3 enhanced the LGR5 promoter activity to upregulate LGR5 expression, which was associated with increased cancer stemness in HCC. Our findings established that HIPK2/PP1Cα/ITCH axis sustains the de-phosphorylation of Dvl3. This post-translational modification of Dvl3 in turn maintains LGR5 expression and enhances the cancer stemness properties in HCC.

Src‐homology 2 domain–containing phosphatase 2 (Shp2) has been reported to play an important role in the maintenance and self‐renewal of embryonic and adult stem cells, but its role in cancer stem cells (CSCs) remains obscure. Herein, we observed high expression of Shp2 in both chemoresistant hepatocellular carcinomas (HCCs) and recurrent HCCs from patients. A remarkable increase of Shp2 was detected in sorted epithelial cell adhesion molecule–positive or cluster of differentiation 133–positive liver CSCs and in CSC‐enriched hepatoma spheroids from patients. Up‐regulated Shp2 facilitated liver CSC expansion by promoting the dedifferentiation of hepatoma cells and enhancing the self‐renewal of liver CSCs. Mechanistically, Shp2 dephosphorylated cell division cycle 73 in the cytosol of hepatoma cells, and the dephosphorylated cell division cycle 73 bound β‐catenin and facilitated the nuclear translocation of β‐catenin, which promoted the dedifferentiation of hepatoma cells. Shp2 increased β‐catenin accumulation by inhibiting glycogen synthase kinase 3β–mediated β‐catenin degradation in liver CSCs, thereby enhancing the self‐renewal of liver CSCs. Blockage of β‐catenin abolished the discrepancy in liver CSC proportion and the self‐renewal capacity between Shp2‐depleted hepatoma cells and control cells, which further confirmed that β‐catenin is required in Shp2‐promoted liver CSC expansion. More importantly, HCC patients with low Shp2 levels benefited from transcatheter arterial chemoembolization or sorafenib treatment, but patients with high Shp2 expression did not, indicating the significance of Shp2 in personalized HCC therapy. Conclusion: Shp2 could promote HCC cell dedifferentiation and liver CSC expansion by amplifying β‐catenin signaling and may be useful in predicting patient response to chemotherapeutics. (Hepatology 2017;65:1566‐1580).

Sorafenib is the only FDA-approved tyrosine kinase inhibitor for targeted therapy in advanced HCC. Nevertheless, its efficacy is limited with only a modest improvement in patient outcome, likely due to acquired resistance. In-depth understanding of the molecular mechanism of sorafenib resistance is warranted for the development of novel treatment strategies. Recent studies by us and others have characterized liver tumor-initiating cells (T-ICs) to be a possible source of resistant and recurrent tumors and a plausible target for HCC treatment. Our group has previously identified CD133 to be a functional marker of liver T-ICs and found annexin a3 (ANXA3) to regulate cancer and stem cell-like properties in this subset of cells. Interestingly, our recent observations also found CD133+ liver T-ICs to be more resistant to sorafenib. Sorafenib resistant clones, established in HepG2 and Huh7 cells by continuous exposure to increasing concentrations of sorafenib, displayed enhanced abilities to migrate, invade, self-renew, and initiate tumor formation in immunodeficient mice, as well as higher expression of stemness associated genes. These two sorafenib resistant cell lines and two other sorafenib resistant HCC patient-derived xenografts established in a similar manner were also found to be enriched for CD133 and ANXA3 expression. Sorafenib resistant clones with ANXA3 stably suppressed were re-sensitized to sorafenib treatment and had diminished ability to migrate, invade, self-renew and initiate tumor growth in vivo, further substantiating the role of ANXA3 in mediating sorafenib resistance in HCC. Mechanistically, an activated PKC/ERK/FRA2 signaling axis was found to be responsible for driving this phenomenon. Clinically, ANXA3 expression was also found to have prognostic value as a higher ANXA3 expression in HCC patients who have received sorafenib treatment was correlated with poor overall survival. The combinatorial use of a homemade ANXA3 neutralizing antibody and sorafenib on HCC patient derived xenografts is now being investigated as a potential new treatment regimen for combating sorafenib resistance in HCC. Citation Format: Man Tong, Steve Luk, Noelia Che, Jin Ding, Terence KW Lee, Stephanie Ma. Targeting ANXA3 in combination with sorafenib for the treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3146. doi:10.1158/1538-7445.AM2017-3146