QSOX1 facilitates dormant esophageal cancer stem cells to evade immune elimination via PD-L1 upregulation and CD8 T cell exclusion.

Dormant cancer stem cells (DCSCs) exhibit characteristics of chemotherapy resistance and immune escape, and they are a crucial source of tumor recurrence and metastasis. However, the underlying mechanisms remain unrevealed. We demonstrate that enriched Gzmk+ CD8+ T cells within the niche of esophageal DCSCs restrict the outgrowth of tumor mass. Nonetheless, DCSCs can escape immune elimination by enhancing PD-L1 signaling, thereby maintaining immune equilibrium. Quiescent fibroblast-derived quiescin sulfhydryl oxidase 1 (QSOX1) promotes the expression of PD-L1 and its own expression in DCSCs by elevating the level of reactive oxygen species. Additionally, high QSOX1 in the dormant tumor niche contributes to the exclusion of CD8+ T cells. Conversely, blocking QSOX1 with Ebselen in combination with anti-PD-1 and chemotherapy can effectively eradicate residual DCSCs by reducing PD-L1 expression and promoting CD8+ T cell infiltration. Clinically, high expression of QSOX1 predicts a poor response to anti-PD-1 treatment in patients with esophageal cancer. Thus, our findings reveal a mechanism whereby QSOX1 promotes PD-L1 upregulation and T cell exclusion, facilitating the immune escape of DCSCs, and QSOX1 inhibition, combined with immunotherapy and chemotherapy, represents a promising therapeutic approach for eliminating DCSCs and preventing recurrence.

Targeting MFGE8 secreted by cancer-associated fibroblasts blocks angiogenesis and metastasis in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could promote cell proliferation, angiogenesis, and ECM reconstitution by binding to integrin families and activating PI3K/AKT pathways in esophageal squamous cell carcinoma (ESCC). The secretions of CAFs play an important role in regulating these biological activities. Among these secretions, we found that MFGE8 is specifically secreted by CAFs in ESCC. Additionally, the secreted MFGE8 protein is essential in CAF-regulated vascularization, tumor proliferation, drug resistance, and metastasis. By binding to Integrin αVß3/αVß5 receptors, MFGE8 promotes tumor progression by activating both the PI3K/AKT and ERK/AKT pathways. Interestingly, the biological function of MFGE8 secreted by CAFs fully demonstrated the major role of CAFs in ESCC and its mode of mechanism, showing that MFGE8 could be a driver factor of CAFs in remodeling the tumor environment. In vivo treatment targeting CAFs-secreting MFGE8 or its receptor produced significant inhibitory effects on ESCC growth and metastasis, which provides an approach for the treatment of ESCC.

ARHGAP15 promotes metastatic colonization in gastric cancer by suppressing RAC1-ROS pathway.

The molecular mechanism of tumor metastasis, especially how metastatic tumor cells colonize in a distant site, remains poorly understood. Here we reported that ARHGAP15, a Rho GTPase activating protein, enhanced gastric cancer (GC) metastatic colonization, which was quite different from its reported role as a tumor suppressor gene in other cancers. It was upregulated in metastatic lymph nodes and significantly associated with a poor prognosis. Ectopic expression of ARHGAP15 promoted metastatic colonization of gastric cancer cells in murine lungs and lymph nodes in vivo or protected cells from oxidative-related death in vitro. However, genetic downregulation of ARHGAP15 had the opposite effect. Mechanistically, ARHGAP15 inactivated RAC1 and then decreased intracellular accumulation of reactive oxygen species (ROS), thus enhancing the antioxidant capacity of colonizing tumor cells under oxidative stress. This phenotype could be phenocopied by inhibition of RAC1 or rescued by the introduction of constitutively active RAC1 into cells. Taken together, these findings suggested a novel role of ARHGAP15 in promoting gastric cancer metastasis by quenching ROS through inhibiting RAC1 and its potential value for prognosis estimation and targeted therapy.

Enhanced mitophagy driven by ADAR1-GLI1 editing supports the self-renewal of cancer stem cells in HCC.

BACKGROUND AND AIMS: Deregulation of adenosine-to-inosine editing by adenosine deaminase acting on RNA 1 (ADAR1) leads to tumor-specific transcriptome diversity with prognostic values for HCC. However, ADAR1 editase-dependent mechanisms governing liver cancer stem cell (LCSC) generation and maintenance have remained elusive. APPROACH AND RESULTS: RNA-seq profiling identified ADAR1-responsive recoding editing events in HCC and showed editing frequency of GLI1 , rather than transcript abundance was clinically relevant. Functional differences in LCSC self-renewal and tumor aggressiveness between wild-type (GLI1 wt ) and edited GLI1 (GLI1 edit ) were elucidated. We showed that overediting of GLI1 induced an arginine-to-glycine (R701G) substitution, augmenting tumor-initiating potential and exhibiting a more aggressive phenotype. GLI1 R701G harbored weak affinity to SUFU, which in turn, promoted its cytoplasmic-to-nuclear translocation to support LCSC self-renewal by increased pluripotency gene expression. Moreover, editing predisposed to stabilize GLI1 by abrogating ß-TrCP-GLI1 interaction. Integrative analysis of single-cell transcriptome further revealed hyperactivated mitophagy in ADAR1-enriched LCSCs. GLI1 editing promoted a metabolic switch to oxidative phosphorylation to control stress and stem-like state through PINK1-Parkin-mediated mitophagy in HCC, thereby conferring exclusive metastatic and sorafenib-resistant capacities. CONCLUSIONS: Our findings demonstrate a novel role of ADAR1 as an active regulator for LCSCs properties through editing GLI1 in the highly heterogeneous HCC.

LOXL1 promotes tumor cell malignancy and restricts CD8 + T cell infiltration in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality globally. Lymph node metastasis and immunosuppression are main factors of poor prognosis in CRC patients. Lysyl oxidase like 1 (LOXL1), part of the lysyl oxidase (LOX) family, plays a yet unclear role in CRC. This study aimed to identify effective biomarkers predictive of prognosis and efficacy of immunotherapy in CRC patients, and to elucidate the prognostic value, clinical relevance, functional and molecular features, and immunotherapy predictive role of LOXL1 in CRC and pan-cancer. METHODS: Weighted gene co-expression network analysis (WGCNA) was employed to explore gene modules related to tumor metastasis and CD8 + T cell infiltration. LOXL1 emerged as a hub gene through differential gene expression and survival analysis. The molecular signatures, functional roles, and immunological characteristics affected by LOXL1 were analyzed in multiple CRC cohorts, cell lines and clinical specimens. Additionally, LOXL1's potential as an immunotherapy response indicator was assessed, along with its role in pan-cancer. RESULTS: Turquoise module in WGCNA analysis was identified as the hub module associated with lymph node metastasis and CD8 + T cell infiltration. Aberrant elevated LOXL1 expression was observed in CRC and correlated with poorer differentiation status and prognosis. Molecular and immunological characterization found that LOXL1 might mediate epithelial-mesenchymal transition (EMT) process and immunosuppressive phenotypes of CRC. Functional study found that LOXL1 enhanced tumor cell proliferation, migration and invasion. Moreover, high LOXL1 levels corresponded to reduced CD8 + T cell infiltration and predicted poor clinical outcomes of immunotherapy. Similar trends were also observed at the pan-cancer level. CONCLUSIONS: Our findings underscore the critical role of LOXL1 in modulating both malignancy and immunosuppression in CRC. This positions LOXL1 as a promising biomarker for predicting prognosis and the response to immunotherapy in CRC patients.

Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos.

OBJECTIVE: Growing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown. DESIGN: The functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10. RESULTS: Functionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial-mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects. CONCLUSION: Altogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

HKDC1 upregulation promotes glycolysis and disease progression, and confers chemoresistance onto gastric cancer.

There is increasing evidence that hexokinase is involved in cell proliferation and migration. However, the function of the hexokinase domain containing protein-1 (HKDC1) in gastric cancer (GC) remains unclear. Immunohistochemistry analysis and big data mining were used to evaluate the correlation between HKDC1 expression and clinical features in GC. In addition, the biological function and molecular mechanism of HKDC1 in GC were studied by in vitro and in vivo assays. Our study indicated that HKDC1 expression was upregulated in GC tissues compared with adjacent nontumor tissues. High expression of HKDC1 was associated with worse prognosis. Functional experiments demonstrated that HKDC1 upregulation promoted glycolysis, cell proliferation, and tumorigenesis. In addition, HKDC1 could enhance GC invasion and metastasis by inducing epithelial-mesenchymal transition (EMT). Abrogation of HKDC1 could effectively attenuate its oncogenic and metastatic function. Moreover, HKDC1 promoted GC proliferation and migration in vivo. HKDC1 overexpression conferred chemoresistance to cisplatin, oxaliplatin, and 5-fluorouracil (5-Fu) onto GC cells. Furthermore, nuclear factor kappa-B (NF-κB) inhibitor PS-341 could attenuate tumorigenesis, metastasis, and drug resistance ability induced by HKDC1 overexpression in GC cells. Our results highlight a critical role of HKDC1 in promoting glycolysis, tumorigenesis, and EMT of GC cells via activating the NF-κB pathway. In addition, HKDC1-mediated drug resistance was associated with DNA damage repair, which further activated NF-κB signaling. HKDC1 upregulation may be used as a potential indicator for choosing an effective chemotherapy regimen for GC patients undergoing chemotherapy.

High Throughput Confined Migration Microfluidic Device for Drug Screening.

Cancer metastasis is the major cause of cancer-related death. Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors, creating confined spaces for tumor cell migration and metastasis. Confined migration is involved in all metastasis steps. However, confined and unconfined migration inhibitors are different and drugs available to inhibit confined migration are rare. The main challenges are the modeling of confined migration, the suffering of low throughput, and others. Microfluidic device has the advantage to reduce reagent consumption and enhance throughput. Here, a microfluidic chip that can achieve multi-function drug screening against the collective migration of cancer cells under confined environment is designed. This device is applied to screen out effective drugs on confined migration among a novel mechanoreceptors compound library (166 compounds) in hepatocellular carcinoma, non-small lung cancer, breast cancer, and pancreatic ductal adenocarcinoma cells. Three compounds that can significantly inhibit confined migration in pan-cancer: mitochonic acid 5 (MA-5), SB-705498, and diphenyleneiodonium chloride are found. Finally, it is elucidated that these drugs targeted mitochondria, actin polymerization, and cell viability, respectively. In sum, a high-throughput microfluidic platform for screening drugs targeting confined migration is established and three novel inhibitors of confined migration in multiple cancer types are identified.

A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtype-specific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy.

Targeting cancer-associated fibroblast-secreted WNT2 restores dendritic cell-mediated antitumour immunity.

OBJECTIVE: Solid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC). DESIGN: Anti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses. RESULTS: A negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades. CONCLUSIONS: CAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

Peritumoral B cells drive proangiogenic responses in HMGB1-enriched esophageal squamous cell carcinoma.

Several B-cell subsets with distinct functions and polarized cytokine profiles that extend beyond antibody production have been reported in different cancers. Here we have demonstrated that proliferating B cells were predominantly found in the peritumoral region of esophageal squamous cell carcinoma (ESCC). These B cells were enriched in tumor nests with high expression of high-mobility group box 1 (HMGB1). High densities of peritumoral proliferating B cells and concomitantly high intratumoral HMGB1 expression showed improved prognostic significance, surpassing prognostic stratification of ESCC patients based on HMGB1 positivity alone. This striking association led us to set up models to test whether cancer-derived HMGB1 could shape tumor microenvironment via modulation on B cells. Overexpression of HMGB1 in ESCC cell lines (KYSE510 and EC18) enhanced proliferation and migration of B cells. Transcriptomic analysis showed that migratory B cells exhibited high enrichment of proangiogenic genes. VEGF expression in proliferating B cells was induced upon co-culture of HMGB1-overexpressing tumor cells and B cells. Secretome array profiling of conditioned media (CM) from the co-culture revealed rich expression of proangiogenic proteins. Consequently, incubation of human umbilical vein endothelial cells with CM promoted angiogenesis in tube formation and migration assays. HMGB1 inhibitor, glycyrrhizin, abolishes all the observed proangiogenic phenotypes. Finally, co-injection of B cells and CM with HMGB1-overexpressing tumor cells, but not with glycyrrhizin, significantly enhanced tumor growth associated with increased microvascular density in ESCC xenograft mice model. Our results indicate that cancer-derived HMGB1 elevates angiogenesis in ESCC by shifting the balance toward proangiogenic signals in proliferating B cells.

Potential synthetic lethality for breast cancer: A selective sirtuin 2 inhibitor combined with a multiple kinase inhibitor sorafenib.

Sorafenib is a clinically useful multiple kinase inhibitor for the treatment of kidney cancer, liver cancer and acute myelocytic leukemia, while it has shown weak efficacy in suppressing breast cancer. Since sirtuin2 (SIRT2) is an important epigenetic regulator and associated with several cancer types including breast cancer, development and evaluation of new SIRT2 inhibitors to probe their therapeutic potentials is currently desirable. A highly selective SIRT2 inhibitor named I was previously developed by us, which showed activity to inhibit non-small cell lung cancer cell lines in vitro. We herein report expanded screening of I and its structurally similar inactive compound II against other cancer cell lines, and found that I had a wide spectrum of anticancer activity while II had no such effects. The I-sorafenib combination treatment exerted obvious synergistic reduction on cell viability of MCF-7 cells. We observed that the combination treatment could suppress cell proliferation, survival and migration, arrest cell cycle at G0/G1 phase, and induce apoptosis in MCF-7 cells, when compared with the single treatment. In vivo studies revealed that the combination treatment showed stronger tumor growth inhibition (87%), comparing with I-(42.8%) or sorafenib-solely-treated groups (61.1%) in MCF-7 xenograft model. In conclusion, this work clearly revealed a potential synthetic lethality effect for I combined with sorafenib, and will probably offer a new strategy at least for breast cancer treatment.

A neural-related gene risk score for head and neck squamous cell carcinoma.

OBJECTIVES: This study aimed to establish a neural-related gene risk score (NRGRS) for the prediction of head and neck squamous cell carcinoma prognosis and explore its predictive value on the benefit of immune checkpoint inhibitor therapy. METHODS: Based on the transcriptome data of HNSCC patients (n = 546) from The Cancer Genome Atlas database, 37 neural-related hub genes were identified by weighted gene co-expression network analysis. Four genes (ITGA5, PYGM, GNG7 and ATP2A3) were identified to construct NRGRS using Lasso-Cox regression method based on the derivation cohort and validated in the Gene Expression Omnibus cohort (n = 109). The survival analysis was performed to validate the prognostic value of NRGRS and immune characteristics in NRGRS-defined subgroups were analyzed. RESULTS: NRGRS-high patients had a worse overall survival than NRGRS-low patients. Tumors with high NRGRS were more likely to have high infiltration of naive CD4+ T cells, M0, M2 macrophages and resting mast cells, which illustrated suppressive immunity and less benefit from immunotherapy therapy. CONCLUSION: NRGRS strongly correlates with survival and is a promising biomarker to predict immunotherapy benefits for head and neck cancer patients. This study provides evidence for the potential correlation between neural-related transcriptome alteration and immune activity.

Cancer Stem Cells in Hepatocellular Carcinoma: Intrinsic and Extrinsic Molecular Mechanisms in Stemness Regulation.

Hepatocellular carcinoma (HCC) remains the most predominant type of liver cancer with an extremely poor prognosis due to its late diagnosis and high recurrence rate. One of the culprits for HCC recurrence and metastasis is the existence of cancer stem cells (CSCs), which are a small subset of cancer cells possessing robust stem cell properties within tumors. CSCs play crucial roles in tumor heterogeneity constitution, tumorigenesis, tumor relapse, metastasis, and resistance to anti-cancer therapies. Elucidation of how these CSCs maintain their stemness features is essential for the development of CSCs-based therapy. In this review, we summarize the present knowledge of intrinsic molecules and signaling pathways involved in hepatic CSCs, especially the CSC surface markers and associated signaling in regulating the stemness characteristics and the heterogeneous subpopulations within the CSC pool. In addition, we recapitulate the effects of crucial extrinsic cellular components in the tumor microenvironment, including stromal cells and immune cells, on the modulation of hepatic CSCs. Finally, we synopsize the currently valuable CSCs-targeted therapy strategies based on intervention in these intrinsic and extrinsic molecular mechanisms, in the hope of shedding light on better clinical management of HCC patients.

Correction: Qian et al. Capsaicin Suppresses Cell Proliferation, Induces Cell Cycle Arrest and ROS Production in Bladder Cancer Cells through FOXO3a-Mediated Pathways. Molecules 2016, 21, 1406.

During the course of a review of our publications, an inadvertent error in the article [...].

PARP inhibitor Olaparib overcomes Sorafenib resistance through reshaping the pluripotent transcriptome in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide with very poor prognosis. Resistance to targeted therapeutic drugs such as sorafenib remains one of the major challenges in clinical treatment. In the present study, PARP1 was found to be highly expressed in human embryonic stem cells, but progressively decreased upon specified hepatic differentiation. Reactivation of PARP1 expression was also detected in HCC residual tumors after sorafenib treatment in xenograft mouse model, indicating the potential important roles of PARP1 in stem cell pluripotency and HCC sorafenib treatment resistance. Overexpression of PARP1 was frequently observed in HCC patients, and closely associated with poor clinical outcome. Treatment of Sorafenib induced activation of DNA damage repair signaling, which is highly active and essential for maintenance of stem cell pluripotency in HCC residual tumors. PARP inhibitor Olaparib extensively suppressed the DNA damage repair signaling, and significantly inhibited the global pluripotent transcriptional network. The repression of key pluripotent transcriptional factors and DNA damage repair signaling by Olaparib was mainly through CHD1L-mediated condensation of the chromatin structure at their promotor regions. The global reshaping of the pluripotent transcriptome by Olaparib might reinforce Sorafenib in eliminating HCC residual tumors and enhance therapeutic efficiency.

TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines.

BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.

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.

Expansion of cancer stem cell pool initiates lung cancer recurrence before angiogenesis.

Angiogenesis is essential in the early stage of solid tumor recurrence, but how a suspensive tumor is reactivated before angiogenesis is mostly unknown. Herein, we stumble across an interesting phenomenon that s.c. xenografting human lung cancer tissues can awaken the s.c. suspensive tumor in nude mice. We further found that a high level of insulin-like growth factor 1 (IGF1) was mainly responsible for triggering the transition from suspensive tumor to progressive tumor in this model. The s.c. suspensive tumor is characterized with growth arrest, avascularity, and a steady-state level of proliferating and apoptotic cells. Intriguingly, CD133+ lung cancer stem cells (LCSCs) are highly enriched in suspensive tumor compared with progressive tumor. Mechanistically, high IGF1 initiates LCSCs self-renewal from asymmetry to symmetry via the activation of a PI3K/Akt/ß-catenin axis. Next, the expansion of LCSC pool promotes angiogenesis by increasing the production of CXCL1 and PlGF in CD133+ LCSCs, which results in lung cancer recurrence. Clinically, a high level of serum IGF1 in lung cancer patients after orthotopic lung cancer resection as an unfavorable factor is strongly correlated with the high rate of recurrence and indicates an adverse progression-free survival. Vice versa, blocking IGF1 or CXCL1/PlGF with neutralizing antibodies can prevent the reactivation of a suspensive tumor induced by IGF1 stimulation in the mouse model. Collectively, the expansion of LCSC pool before angiogenesis induced by IGF1 is a key checkpoint during the initiation of cancer relapse, and targeting serum IGF1 may be a promising treatment for preventing recurrence in lung cancer patients.