SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma.

Tumor lineage plasticity, considered a hallmark of cancer, denotes the phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the underlying molecular events remain largely elusive. Our recent study identified CD133/Prom1 in hepatocellular carcinoma (HCC) tumors to mark proliferative tumor-propagating cells with cancer stem cell-like properties, that follow a dedifferentiation trajectory towards a more embryonic state. Here we show SPINK1 to strongly associate with CD133 + HCC, and tumor dedifferentiation. Enhanced transcriptional activity of SPINK1 is mediated by promoter binding of ELF3, which like CD133, is found to increase following 5-FU and cisplatin treatment; while targeted depletion of CD133 will reduce both ELF3 and SPINK1. Functionally, SPINK1 overexpression promotes tumor initiation, self-renewal, and chemoresistance by driving a deregulated EGFR-ERK-CDK4/6-E2F2 signaling axis to induce dedifferentiation of HCC cells into their ancestral lineages. Depleting SPINK1 function by neutralizing antibody treatment or in vivo lentivirus-mediated Spink1 knockdown dampens HCC cancer growth and their ability to resist chemotherapy. Targeting oncofetal SPINK1 may represent a promising therapeutic option for HCC treatment.

SERPINA12 promotes the tumorigenic capacity of HCC stem cells through hyperactivation of AKT/ß-catenin signaling.

BACKGROUND AND AIMS: HCC is an aggressive disease with poor clinical outcome. Understanding the mechanisms that drive cancer stemness, which we now know is the root cause of therapy failure and tumor recurrence, is fundamental for designing improved therapeutic strategies. This study aims to identify molecular players specific to CD133 + HCC to better design drugs that can precisely interfere with cancer stem cells but not normal stem cell function. APPROACH AND RESULTS: Transcriptome profiling comparison of epithelial-specific "normal" CD133 + cells isolated from fetal and regenerating liver against "HCC" CD133 + cells isolated from proto-oncogene-driven and inflammation-associated HCC revealed preferential overexpression of SERPINA12 in HCC but not fetal and regenerating liver CD133 + cells. SERPINA12 upregulation in HCC is tightly associated with aggressive clinical and stemness features, including survival, tumor stage, cirrhosis, and stemness signatures. Enrichment of SERPINA12 in HCC is mediated by promoter binding of the well-recognized ß-catenin effector TCF7L2 to drive SERPINA12 transcriptional activity. Functional characterization identified a unique and novel role of endogenous SERPINA12 in promoting self-renewal, therapy resistance, and metastatic abilities. Mechanistically, SERPINA12 functioned through binding to GRP78, resulting in a hyperactivated AKT/GSK3ß/ß-catenin signaling cascade, forming a positive feed-forward loop. Intravenous administration of rAAV8-shSERPINA12 sensitized HCC cells to sorafenib and impeded the cancer stem cell subset in an immunocompetent HCC mouse model. CONCLUSIONS: Collectively, our findings revealed that SERPINA12 is preferentially overexpressed in epithelial HCC CD133 + cells and is a key contributor to HCC initiation and progression by driving an AKT/ß-catenin feed-forward loop.

Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-ß Signaling.

BACKGROUND & AIMS: Metastasis is found in most advanced hepatocellular carcinoma (HCC) patients, and it drives tumor recurrence and systemic failure. There is no effective treatment owing to its complex biological features. Many of the molecular drivers of metastasis are crucial players in normal physiology but behave unconventionally during cancer progression. Targeting these molecular drivers for therapy and differentiating them from a physiological background require a detailed examination of the novel mechanisms involved in their activation during metastasis. METHODS: Publicly available transcriptomic data such as that of TCGA-LIHC and Gene Expression Omnibus were utilized to identify novel targets upregulated in advanced and metastatic HCC. Validation of candidates was assisted by immunohistochemistry performed on tissue microarrays derived from more than 100 HCC patients. Expression of protein tyrosine kinase 7 (PTK7) was studied under the treatment of transforming growth factor-ß1 and knockdown of SRY-Box Transcription Factor 9 (SOX9) to delineate upstream regulation, while CRISPR-mediated knockout and lentiviral overexpression of PTK7 in HCC cells were performed to study their functional and signaling consequences. Manipulated HCC cells were injected into mice models either by orthotopic or tail-vein injection to observe for any in vivo pro-metastatic effects. RESULTS: PTK7 was discovered to be the kinase most significantly upregulated in advanced and metastatic HCC, at both transcriptomic and proteomic level. Bioinformatic analyses and functional assays performed in HCC cell lines revealed transforming growth factor-ß signaling and SOX9 to be important activators of PTK7 expression. Functionally, enrichment of PTK7 expression could positively regulate metastatic potential of HCC cells in vitro and in lung metastasis models performed in immunodeficient mice. The up-regulation of PTK7 recruited the epithelial-mesenchymal transition components, zinc finger protein SNAI2 (SLUG) and zinc finger E-box-binding homeobox 1 (ZEB1). CONCLUSIONS: Our study proposes PTK7 as a novel molecular driver in metastatic HCC, particularly in a transforming growth factor-ß-activated microenvironment. The preferential expression of PTK7 resulted in a previously unobserved regulatory effect on the recruitment of epithelial-mesenchymal transition components, which established PTK7 as a potential determinant of specific epithelial-mesenchymal transition status. Therefore, our data support the continual development of PTK7-targeted agents as antimetastatic therapies.

Repurposing hyperpolarization-activated cyclic nucleotide-gated channels as a novel therapy for breast cancer.

Hyperpolarization-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.

FSTL1 Secreted by Activated Fibroblasts Promotes Hepatocellular Carcinoma Metastasis and Stemness.

The tumor microenvironment plays a critical role in maintaining the immature phenotype of tumor-initiating cells (TIC) to promote cancer. Hepatocellular carcinoma (HCC) is a unique disease in that it develops in the setting of fibrosis and cirrhosis. This pathologic state commonly shows an enrichment of stromal myofibroblasts, which constitute the bulk of the tumor microenvironment and contribute to disease progression. Follistatin-like 1 (FSTL1) has been widely reported as a proinflammatory mediator in different fibrosis-related and inflammatory diseases. Here we show FSTL1 expression to be closely correlated with activated fibroblasts and to be elevated in regenerative, fibrotic, and disease liver states in various mouse models. Consistently, FSTL1 lineage cells gave rise to myofibroblasts in a CCL4-induced hepatic fibrosis mouse model. Clinically, high FSTL1 in fibroblast activation protein-positive (FAP+) fibroblasts were significantly correlated with more advanced tumors in patients with HCC. Although FSTL1 was expressed in primary fibroblasts derived from patients with HCC, it was barely detectable in HCC cell lines. Functional investigations revealed that treatment of HCC cells and patient-derived 3D organoids with recombinant FSTL1 or with conditioned medium collected from hepatic stellate cells or from cells overexpressing FSTL1 could promote HCC growth and metastasis. FSTL1 bound to TLR4 receptor, resulting in activation of AKT/mTOR/4EBP1 signaling. In a preclinical mouse model, blockade of FSTL1 mitigated HCC malignancy and metastasis, sensitized HCC tumors to sorafenib, prolonged survival, and eradicated the TIC subset. Collectively, these data suggest that FSTL1 may serve as an important novel diagnostic/prognostic biomarker and therapeutic target in HCC. SIGNIFICANCE: This study shows that FSTL1 secreted by activated fibroblasts in the liver microenvironment augments hepatocellular carcinoma malignancy, providing a potential new strategy to improve treatment of this aggressive disease.

EPHB2 Activates ß-Catenin to Enhance Cancer Stem Cell Properties and Drive Sorafenib Resistance in Hepatocellular Carcinoma.

The survival benefit derived from sorafenib treatment for patients with hepatocellular carcinoma (HCC) is modest due to acquired resistance. Targeting cancer stem cells (CSC) is a possible way to reverse drug resistance, however, inhibitors that specifically target liver CSCs are limited. In this study, we established two sorafenib-resistant, patient-derived tumor xenografts (PDX) that mimicked development of acquired resistance to sorafenib in patients with HCC. RNA-sequencing analysis of sorafenib-resistant PDXs and their corresponding mock controls identified EPH receptor B2 (EPHB2) as the most significantly upregulated kinase. EPHB2 expression increased stepwise from normal liver tissue to fibrotic liver tissue to HCC tissue and correlated with poor prognosis. Endogenous EPHB2 knockout showed attenuation of tumor development in mice. EPHB2 regulated the traits of liver CSCs; similarly, sorted EPHB2High HCC cells were endowed with enhanced CSC properties when compared with their EPHB2-Low counterparts. Mechanistically, EPHB2 regulated cancer stemness and drug resistance by driving the SRC/AKT/GSK3ß/ß-catenin signaling cascade, and EPHB2 expression was regulated by TCF1 via promoter activation, forming a positive Wnt/ß-catenin feedback loop. Intravenous administration of rAAV-8-shEPHB2 suppressed HCC tumor growth and significantly sensitized HCC cells to sorafenib in an NRAS/AKT-driven HCC immunocompetent mouse model. Targeting a positive feedback loop involving the EPHB2/ß-catenin axis may be a possible therapeutic strategy to combat acquired drug resistance in HCC. SIGNIFICANCE: This study identifies a EPHB2/ß-catenin/TCF1 positive feedback loop that augments cancer stemness and sorafenib resistance in HCC, revealing a targetable axis to combat acquired drug resistance in HCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3229/F1.large.jpg.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

Chemotherapy-Enriched THBS2-Deficient Cancer Stem Cells Drive Hepatocarcinogenesis through Matrix Softness Induced Histone H3 Modifications.

The physical microenvironment is a critical mediator of tumor behavior. However, detailed biological and mechanistic insight is lacking. The present study reveals the role of chemotherapy-enriched CD133+ liver cancer stem cells (CSCs) with THBS2 deficiency. This subpopulation of cells contributes to a more aggressive cancer and functional stemness phenotype in hepatocellular carcinoma (HCC) by remodeling the extracellular matrix (ECM) through the regulation of matrix metalloproteinase (MMP) activity, collagen degradation, and matrix stiffness. The local soft spots created by these liver CSCs can enhance stemness and drug resistance and provide a route of escape to facilitate HCC metastasis. Interestingly, a positive feed-forward loop is identified where a local soft spot microenvironment in the HCC tumor is enriched with CD133 expressing cells that secrete markedly less ECM-modifying THBS2 upon histone H3 modification at its promoter region, allowing the maintenance of a localized soft spot matrix. Clinically, THBS2 deficiency is also correlated with low HCC survival, where high levels of CSCs with low THBS2 expression in HCC are associated with decreased collagen fiber deposits and an invasive tumor front. The findings have implications for the treatment of cancer stemness and for the prevention of tumor outgrowth through disseminated tumor cells.

PRMT6 deficiency induces autophagy in hostile microenvironments of hepatocellular carcinoma tumors by regulating BAG5-associated HSC70 stability.

Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis by degrading damaged organelles and unwanted proteins and supports cellular biosynthesis in response to stress. Cancer cells, including hepatocellular carcinoma (HCC), are often situated in a hypoxic, nutrient-deprived and stressful microenvironment where tumor cells are yet still able to adapt and survive. However, the mechanism underlying this adaptation and survival is not well-defined. We report deficiency of the post-translational modification enzyme protein arginine N-methyltransferase 6 (PRMT6) in HCC to promote the induction of autophagy under oxygen/nutrient-derived and sorafenib drug-induced stress conditions. Enhanced autophagic flux in HCC cells negatively correlated with PRMT6 expression, with the catalytic domain of PRMT6 critically important in mediating these autophagic activities. Mechanistically, PRMT6 physically interacts and methylates BAG5 to enhance the degradation of its interacting partner HSC70, a well-known autophagy player. The therapeutic potential of targeting BAG5 using genetic approach to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC is also demonstrated in an in vivo model. The clinical implications of these findings are highlighted by the inverse correlative expressions of PRMT6 and HSC70 in HCC tissues. Collectively, deficiency of PRMT6 induces autophagy to promote tumorigenicity and cell survival in hostile microenvironments of HCC tumors by regulating BAG5-associated HSC70 stability through post-translational methylation of BAG5. Targeting BAG5 may therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to sorafenib for treatment.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

BACKGROUND AND AIMS: Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. APPROACH AND RESULTS: Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. CONCLUSIONS: Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

CRAF Methylation by PRMT6 Regulates Aerobic Glycolysis-Driven Hepatocarcinogenesis via ERK-Dependent PKM2 Nuclear Relocalization and Activation.

BACKGROUND AND AIMS: Most tumor cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and promote tumorigenicity and drug resistance. Intriguingly, the molecular mechanisms underlying this phenomenon are not well understood. In this work, using gain-of-function and loss-of-function in vitro studies in patient-derived organoid and cell cultures as well as in vivo positron emission tomography-magnetic resonance imaging animal models, we showed that protein arginine N-methyltransferase 6 (PRMT6) regulates aerobic glycolysis in human hepatocellular carcinoma (HCC) through nuclear relocalization of pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. APPROACH AND RESULTS: We found PRMT6 to methylate CRAF at arginine 100, interfering with its RAS/RAF binding potential, and therefore altering extracellular signal-regulated kinase (ERK)-mediated PKM2 translocation into the nucleus. This altered PRMT6-ERK-PKM2 signaling axis was further confirmed in both a HCC mouse model with endogenous knockout of PRMT6 as well as in HCC clinical samples. We also identified PRMT6 as a target of hypoxia through the transcriptional repressor element 1-silencing transcription factor, linking PRMT6 with hypoxia in driving glycolytic events. Finally, we showed as a proof of concept the therapeutic potential of using 2-deoxyglucose, a glycolysis inhibitor, to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC. CONCLUSIONS: Our findings indicate that the PRMT6-ERK-PKM2 regulatory axis is an important determinant of the Warburg effect in tumor cells, and provide a mechanistic link among tumorigenicity, sorafenib resistance, and glucose metabolism.

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

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 downregulated in hepatocellular carcinoma (HCC) 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 cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6-/-) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100.

Efficacy of annexin A3 blockade in sensitizing hepatocellular carcinoma to sorafenib and regorafenib.

BACKGROUND & AIMS: Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Sorafenib is the only FDA-approved first-line targeted drug for advanced HCC, but its effect on patient survival is limited. Further, patients ultimately present with disease progression. A better understanding of the causes of sorafenib resistance, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCC. METHODS: The functional effects of ANXA3 in conferring sorafenib resistance to HCC cells were analyzed in apoptotic and tumorigenicity assays. The role of ANXA3/PKCδ-mediated p38 signaling, and subsequently altered autophagic and apoptotic events, was assessed by immunoprecipitation, immunoblotting, immunofluorescence and transmission electron microscopy assays. The prognostic value of ANXA3 in predicting response to sorafenib was evaluated by immunohistochemistry. The therapeutic value of targeting ANXA3 to combat HCC with anti-ANXA3 monoclonal antibody alone or in combination with sorafenib/regorafenib was investigated ex vivo and in vivo. RESULTS: ANXA3 conferred HCC cells with resistance to sorafenib. ANXA3 was found enriched in sorafenib-resistant HCC cells and patient-derived xenografts. Mechanistically, overexpression of ANXA3 in sorafenib-resistant HCC cells suppressed PKCδ/p38 associated apoptosis and activated autophagy for cell survival. Clinically, ANXA3 expression correlated positively with the autophagic marker LC3B in HCC and was associated with a worse overall survival in patients who went on to receive sorafenib treatment. Anti-ANXA3 monoclonal antibody therapy combined with sorafenib/regorafenib impaired tumor growth in vivo and significantly increased survival. CONCLUSION: Anti-ANXA3 therapy in combination with sorafenib/regorafenib represents a novel therapeutic strategy for HCC treatment. ANXA3 represents a useful predictive biomarker to stratify patients with HCC for sorafenib treatment. LAY SUMMARY: This study represents the most extensive pre-clinical characterization of anti-ANXA3 monoclonal antibodies for the treatment of hepatocellular carcinoma to date. These results support the clinical trial development of anti-ANXA3 antibodies in combination with sorafenib/regorafenib. Further studies will optimize patient target selection and identify the best treatment combinations.

IRAK1 Augments Cancer Stemness and Drug Resistance via the AP-1/AKR1B10 Signaling Cascade in Hepatocellular Carcinoma.

Frequent relapse and drug resistance in patients with hepatocellular carcinoma (HCC) can be attributed to the existence of tumor-initiating cells (TIC) within the tumor bulk. Therefore, targeting liver TICs may improve the prognosis of these patients. From transcriptome sequencing of 16 pairs of clinical HCC samples, we report that interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway is significantly upregulated in HCC. IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patient survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also consistently upregulated. IRAK1 regulated liver TIC properties, including self-renewal, tumorigenicity, and liver TIC marker expression. IRAK1 inhibition sensitized HCC cells to doxorubicin and sorafenib treatment in vitro via suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver TIC populations. We identified aldo-keto reductase family 1 member 10 (AKR1B10) as a novel downstream target of IRAK1, which was found to be overexpressed in HCC and significantly correlated with IRAK1 expression. Knockdown of AKR1B10 negated IRAK1-induced TIC functions via modulation of the AP-1 complex. Inhibition of IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed tumor growth in an HCC xenograft model. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC.Significance: IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway regulates cancer stemness and drug resistance and may be a novel therapeutic target in HCC. Cancer Res; 78(9); 2332-42. ©2018 AACR.

BQ323636.1, a Novel Splice Variant to NCOR2, as a Predictor for Tamoxifen-Resistant Breast Cancer.

Purpose: 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 AACRSee related commentary by Jordan, p. 3480.

FSTL1 Promotes Metastasis and Chemoresistance in Esophageal Squamous Cell Carcinoma through NFκB-BMP Signaling Cross-talk.

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886-99. ©2017 AACR.

TP53INP1 Downregulation Activates a p73-Dependent DUSP10/ERK Signaling Pathway to Promote Metastasis of Hepatocellular Carcinoma.

Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. Cancer Res; 77(17); 4602-12. ©2017 AACR.

Notch Inhibitor PF-03084014 Inhibits Hepatocellular Carcinoma Growth and Metastasis via Suppression of Cancer Stemness due to Reduced Activation of Notch1-Stat3.

Aberrant activation of the Notch signaling pathway is implicated in many solid tumors, including hepatocellular carcinoma, indicating a potential use of Notch inhibitors for treatment. In this study, we investigated the antitumor and antimetastasis efficacy of the novel Notch inhibitor (γ-secretase inhibitor) PF-03084014 in hepatocellular carcinoma. Hepatocellular carcinoma spherical cells (stem-like cancer cells), a sphere-derived orthotopic tumor model and one patient-derived xenograft (PDX) model were used in our experiment. We demonstrated that PF-03084014 inhibited the self-renewal and proliferation of cancer stem cells. PF-03084014 reduced the hepatocellular carcinoma sphere-derived orthotopic tumor and blocked the hepatocellular carcinoma tumor liver to lung metastasis. We further tested the PF-03084014 in PDX models and confirmed the inhibition tumor growth effect. In addition, a low dose of PF-03084014 induced hepatocellular carcinoma sphere differentiation, resulting in chemosensitization. Antitumor activity was associated with PF-03084014-induced suppression of Notch1 activity, decreased Stat3 activation and phosphorylation of the Akt signaling pathway, and reduced epithelial-mesenchymal transition. These are the key contributors to the maintenance of cancer stemness and the promotion of cancer metastasis. Moreover, the Notch-Stat3 association was implicated in the clinical hepatocellular carcinoma prognosis. Collectively, PF-03084014 revealed antitumor and antimetastatic effects in hepatocellular carcinoma, providing evidence for the potential use of gamma-secretase inhibitors as a therapeutic option for the treatment of hepatocellular carcinoma. Mol Cancer Ther; 16(8); 1531-43. ©2017 AACR.

Octamer 4/microRNA-1246 signaling axis drives Wnt/ß-catenin activation in liver cancer stem cells.

Wnt/ß-catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR-1246, promotes cancer stemness, including self-renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/ß-catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3ß (GSK3ß), two key members of the ß-catenin destruction complex. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR-1246, which cooperatively drive ß-catenin activation in liver CSCs. CONCLUSION: These findings uncover the noncanonical regulation of Wnt/ß-catenin in liver CSCs by the Oct4/miR-1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062-2076).