Prognostic significance of SASP-related gene signature of radiation therapy in head and neck squamous cell carcinoma.

In this study,we developed and validated the clinical significance of senescence SASP related gene signature and explored its association with radiation therapy (RT) in patients with head and neck squamous cell carcinoma (HNSCC). First, we searched the three published review literature associated with senescence associated secretory phenotype (SASP) and selected all 81 genes to develop SASP related gene signature. Then, 81 SASP related genes were adapted to gene expression dataset from TCGA. HNSCC patients of TCGA were classified into clusters 1 and 2 via unsupervised clustering according to SASP related gene signature. Kaplan-Meier plot survival analysis showed that cluster 1 had a poorer prognosis than cluster 2 in 5-years overall survival and recurrence-free survival. Similarly, cluster 1 showed a worse prognosis than cluster 2 in three validation cohorts. (E-MTAB-8588, FHCRC and KHU). Cox proportional hazards regression observed that the senescence SASP related signature was an independent prognostic factor for HNSCC patients. We also established a nomogram using a relevant clinical parameter and a risk score. Time-dependent receiver operating characteristic (ROC) analysis was carried out to assess the accuracy of the prognostic risk model and nomogram. Senescence SASP related gene signature was associated with the response to RT. Therefore, subsequent, in vitro experiments further validated the association between senescence SASP related gene signature and RT in HNSCC. In conclusion, we developed a senescence SASP related gene signature, which could predict survival of HNSCC patients, and this gene signature provides new clinical evidence for the accurate diagnosis and targeted RT of HNSCC.

Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Cell Death Induced by the Combination of Ephedra sinica Extract and Radiation in HNSCC is Positively Related to BAX and p-MLKL Expression.

BACKGROUND: Numerous studies have proven the efficacy and safety of natural products, and are widely used as attractive cancer treatments. The investigation of effective natural products for improving cancer treatment is a promising strategy. Combination treatment with radiosensitizers and radiotherapy (RT) is considered necessary for therapeutic improvement in head and neck squamous cell carcinoma(HNSCC). OBJECTIVE: This study aims to investigate whether Ephedra sinica (ES) extract could induce selective cell death in cancer cells and serve as a radiosensitizer for HNSCC. METHODS: HNSCC cells were pretreated with ES extract before radiation, and the radiosensitizing activity was assessed using a colony formation assay. Radiation-induced cell death was evaluated using an annexinV-FITC assay. Western blotting was performed to confirm cell death-related gene expression, including apoptosis and necrosis markers. RESULTS: ES extract significantly inhibited HNSCC cell viability (FaDu and SNU1076), while having minimal effect on normal HaCaT cells. When HNSCC cells were irradiated with 2, 4, or 8 Gy and cultured with ES extract (25 µg/mL), they exhibited increased radiation sensitivity compared to non-treated cells. The combination of ES extract and radiation resulted in increased cell death compared to non-treated, ES-treated, or irradiated cells. The apoptosis marker BAX and necrosis marker p-MLKL expression levels were also elevated following the combination treatment. CONCLUSION: ES extract demonstrated significant cytotoxic potential in HNSCC cells without affecting normal cells. It enhanced the radiosensitivity of HNSCC cells by upregulating BAX and p-MLKL expression, leading to increased cell death. These results suggest ES extract exhibits a potential radiosensitizing capacity in HNSCC.

NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition.

BACKGROUND: Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited. METHODS: In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown. RESULTS: Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation. CONCLUSIONS: Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

Clinically conserved genomic subtypes of gastric adenocarcinoma.

Gastric adenocarcinoma (GAC) is a lethal disease characterized by genomic and clinical heterogeneity. By integrating 8 previously established genomic signatures for GAC subtypes, we identified 6 clinically and molecularly distinct genomic consensus subtypes (CGSs). CGS1 have the poorest prognosis, very high stem cell characteristics, and high IGF1 expression, but low genomic alterations. CGS2 is enriched with canonical epithelial gene expression. CGS3 and CGS4 have high copy number alterations and low immune reactivity. However, CGS3 and CGS4 differ in that CGS3 has high HER2 activation, while CGS4 has high SALL4 and KRAS activation. CGS5 has the high mutation burden and moderately high immune reactivity that are characteristic of microsatellite instable tumors. Most CGS6 tumors are positive for Epstein Barr virus and show extremely high levels of methylation and high immune reactivity. In a systematic analysis of genomic and proteomic data, we estimated the potential response rate of each consensus subtype to standard and experimental treatments such as radiation therapy, targeted therapy, and immunotherapy. Interestingly, CGS3 was significantly associated with a benefit from chemoradiation therapy owing to its high basal level of ferroptosis. In addition, we also identified potential therapeutic targets for each consensus subtype. Thus, the consensus subtypes produced a robust classification and provide for additional characterizations for subtype-based customized interventions.

Gene signature predicting recurrence in oral squamous cell carcinoma is characterized by increased oxidative phosphorylation.

Although numerous studies have used systemic approaches to identify prognostic predictors in oral squamous cell carcinoma (OSCC), the effectiveness of these approaches has not been assessed clinically. Further, the mechanism underlying malignant behaviors in OSCC is poorly characterized. This study aimed to develop and verify accurate prognostic predictors for OSCC patients and assess the associated biology. We identified an OSCC-recurrence-related gene signature (ORGS) using a Cox regression analysis. Functional enrichment analysis was used to identify enriched pathways and biological processes to reveal the underlying mechanism of OSCC malignant behavior. The ORGS successfully divided OSCC patients into low- and high-risk groups with significantly different overall survivals. Pathway analysis revealed oxidative phosphorylation (OXPHOS) as a signaling pathway associated with the ORGS in OSCC. Interestingly, high OXPHOS status was strongly associated with poor overall survival in OSCC patients. Mediator complex subunit 30 (MED30) was a predicted upstream regulator of OXPHOS, and knockdown of MED30 reduced histone acetylation. We identified that the ORGS was strongly correlated with OXPHOS regulatory processes, suggesting OXPHOS as a key mechanism leading to poor prognosis in OSCC.

Association between cancer stem cell gene expression signatures and prognosis in head and neck squamous cell carcinoma.

BACKGROUND: Various cancer stem cell (CSC) biomarkers and the genes encoding them in head and neck squamous cell carcinoma (HNSCC) have been identified and evaluated. However, the validity of these factors in the prognosis of HNSCC has been questioned and remains unclear. In this study, we examined the clinical significance of CSC biomarker genes in HNSCC, using five publicly available HNSCC cohorts. METHODS: To predict the prognosis of patients with HNSCC, we developed and validated the expression signatures of CSC biomarker genes whose mRNA expression levels correlated with at least one of the four CSC genes (CD44, MET, ALDH1A1, and BMI1). RESULTS: Patients in The Cancer Genome Atlas (TCGA) HNSCC cohort were classified into CSC gene expression-associated high-risk (CSC-HR; n = 285) and CSC gene expression-associated low-risk (CSC-LR; n = 281) subgroups. The 5-year overall survival and recurrence-free survival rates were significantly lower in the CSC-HR subgroup than in the CSC-LR subgroup (p = 0.04 and 0.02, respectively). The clinical significance of the CSC gene expression signature was validated using four independent cohorts. Analysis using Cox proportional hazards models showed that the CSC gene expression signature was an independent prognostic factor of non-oropharyngeal HNSCC which mostly indicates HPV (-) status. Furthermore, the CSC gene expression signature was associated with the prognosis of HNSCC patients who received radiotherapy. CONCLUSION: The CSC gene expression signature is associated with the prognosis of HNSCC and may help in personalized treatments for patients with HNSCC, especially in cases with HPV (-) status who were classified in more detail.

Prognostic Significance of the BIRC2-BIRC3 Gene Signature in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) has poor prognosis, with survival rates that have not significantly improved over the past several decades. Therefore, prediction of HNSCC prognosis is of clinical importance. Baculoviral IAP Repeat containing 2 (BIRC2) and Baculoviral IAP Repeat containing 3 (BIRC3) are involved in oncogenic activity by modulating cell proliferation, apoptosis and invasion in HNSCC. This study aimed to develop and validate a predictive gene signature for BIRC2 and BIRC3. MATERIALS AND METHODS: The genomic copy number and gene expression for BIRC2 and BIRC3 were systematically explored in patients with HNSCC to investigate the clinical relevance of BIRC2 and BIRC3 activation. A prognostic signature was developed based on correlations associated with BIRC2 and BIRC3 mRNA expression and copy number alterations. Hierarchical clustering was used to classify the clusters (Clusters 1 and 2). Moreover, independent validation of the BIRC2-BIRC3 gene signature was performed using the Leipzig, MDACC, FHCRC, and KHU datasets. To explore the biological functions of the BIRC2-BIRC3 gene signature, string analysis and pathway annotation were also performed. RESULTS: BIRC2-BIRC3 gene signature-derived cluster 2 patients exhibited significantly poor survival. This signature also predicted survival in three independent cohorts. Interestingly, the BIRC2-BIRC3 gene signature additionally permitted the identification of survival in advanced tumor stages with excellent accuracy in all three cohorts. Multivariate Cox regression analysis identified that the BIRC2-BIRC3 signature was an independent predictor associated with the survival of patients with HNSCC. Moreover, Inhibition of BIRC2 modulated the NF-B signaling pathway via upregulation of CBR1 expression. CONCLUSION: The BIRC2-BIRC3 gene signature was found to be associated with the prognosis of HNSCC. Thus, BIRC2 and BIRC3 could be potential targets for improving HNSCC prognosis.

NANOG confers resistance to complement-dependent cytotoxicity in immune-edited tumor cells through up-regulating CD59.

Cancer immunoediting drives the adaptation of tumor cells to host immune surveillance. Previously, we have demonstrated that immunoediting driven by cytotoxic T lymphocytes (CTLs) enriches NANOG+ tumor cells with immune-refractory properties. Here, we found that CTL-mediated immune pressure triggered cross-resistance of tumor cells to the complement system, a part of the innate immune system. In this process, NANOG upregulated the membrane-bound complement regulatory protein (mCRP) CD59 through promoter occupancy, thereby contributing to the resistance of tumor cells against complement-dependent cytotoxicity (CDC). Notably, targeting of NANOG sensitized the immune-refractory tumor cells to trastuzumab-mediated CDC. Collectively, our results revealed a possible mechanism through which selection imposed by T-cell based immunotherapy triggered complement-resistant phenotypes in the tumor microenvironment (TME), by establishing a firm molecular link between NANOG and CD59 in immune-edited tumor cells. We believe these results hold important implications for the clinical application of CDC-mediated therapeutic antibody.

Clinical significance of FAT1 gene mutation and mRNA expression in patients with head and neck squamous cell carcinoma.

The FAT1 gene functions as a tumor suppressor or promoter and remains incompletely understood. We examined the clinical significance of FAT1 in head and neck squamous cell carcinoma (HNSCC) using four publicly available HNSCC cohorts and one HNSCC cohort enrolled at a tertiary medical center. We developed FAT1 signatures reflecting FAT1 mutations and mRNA expression using one cohort. Patients with HNSCC were classified into FAT1-associated low risk (FAT1-LR; n = 195) and FAT1-associated high risk (FAT1-HR; n = 371) subgroups. The five-year overall survival and recurrence-free survival rates were significantly lower in the FAT1-HR subgroup than in the FAT1-LR subgroup (P = 0.01 and 0.003, respectively). The clinical significance of FAT1 was validated using four independent cohorts. Cox proportional hazards models showed that the FAT1 signature was an independent prognostic factor for HNSCC patients. In addition, FAT1 signature was associated with the response to radiotherapy, advanced stage, and human papilloma virus (HPV) status in HNSCC patients. In conclusion, the FAT1 gene signature was associated with prognosis of HNSCC and may help to provide personalized treatments for HNSCC patients.

Changes of Pepsin Concentration in Saliva Sample According to Storage Period.

OBJECTIVES: To determine whether the concentration of pepsin in the saliva sample changes according to the storage period of the sample. METHODS: Forty eight patients with suspected laryngopharyngeal reflux were included in this study. Saliva samples were collected from each patient and each sample divided into six and stored for different period of time. Pepsin concentration was measured using enzyme-linked immunosorbent assay. A comparison was made between the pepsin concentration measured immediately and the concentration measured after storage for 1 week, 2 weeks, 1 month, 3 months, and 6 months. RESULTS: No significant difference in pepsin concentrations were detected between the sample analyzed immediately and those analyzed at 1 week, 2 weeks, 1 month, and 3 months after saliva collection. A significant difference in pepsin concentration was observed in the sample analyzed immediately and the sample analyzed 6 months after saliva collection. CONCLUSIONS: Pepsin concentration in saliva samples did not demonstrate a significant difference between the concentration measured immediately and the concentration measured 3 months after saliva collection, although a significant difference was observed in the concentration measured 6 months after collection.

SOD2- and NRF2-associated Gene Signature to Predict Radioresistance in Head and Neck Cancer.

BACKGROUND: We propose a novel prognostic biomarker-based strategy for increasing the efficacy of radiotherapy (RT) in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We identified genes associated with superoxide dismutase 2 (SOD2) and nuclear factor erythroid-2-related factor 2 (NRF2) from gene-expression data of The Cancer Genome Atlas (TCGA) by calculating Pearson correlation. Patients were divided into two groups using hierarchical clustering. Colony-formation assay was performed to determine radioresistance in HNSCC cell line CAL27. Pathway analysis was conducted using The Database for Annotation, Visualization and Integrated Discovery (DAVID). RESULTS: We developed a 49-gene signature with SOD2- and NRF2-associated genes. Using mRNA expression data for the 49-gene signature, we performed hierarchical clustering to stratify patients into two subtypes, subtype A and B. In the TCGA cohort, subgroup A demonstrated a better prognosis than subgroup B in patients who received RT. The signature robustness was evaluated in other independent cohorts. We showed through colony-formation assay that depletion of SOD2 or NRF2 leads to increased radiosensitivity. CONCLUSION: We identified and validated a robust gene signature of SOD2- and NRF2-associated genes in HNSCC and confirmed their link to radioresistance using in vitro assay, providing a novel biomarker for the evaluation of HNSCC prognosis.

LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells.

Immune selection drives tumor cells to acquire refractory phenotypes. We previously demonstrated that cytotoxic T lymphocyte (CTL)-mediated immune pressure enriches NANOG+ tumor cells with stem-like and immune-refractory properties that make them resistant to CTLs. Here, we report that the emergence of refractory phenotypes is highly associated with an aberrant macroautophagic/autophagic state of the NANOG+ tumor cells and that the autophagic phenotype arises through transcriptional induction of MAP1LC3B/LC3B by NANOG. Furthermore, we found that upregulation of LC3B expression contributes to an increase in EGF secretion. The subsequent hyperactivation of EGFR-AKT signaling rendered NANOG+ tumor cells resistant to CTL killing. The NANOG-LC3B-p-EGFR axis was preserved across various types of human cancer and correlated negatively with the overall survival of cervical cancer patients. Inhibition of LC3B in immune-refractory tumor models rendered tumors susceptible to adoptive T-cell transfer, as well as PDCD1/PD-1 blockade, and led to successful, long-term control of the disease. Thus, our findings demonstrate a novel link among immune-resistance, stem-like phenotypes, and LC3B-mediated autophagic secretion in immune-refractory tumor cells, and implicate the LC3B-p-EGFR axis as a central molecular target for controlling NANOG+ immune-refractory cancer.Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A1; CASP3: caspase 3; CFSE: carboxyfluorescein succinimidyl ester; ChIP: chromatin immunoprecipitation; CI: confidence interval; CIN: cervical intraepithelial neoplasia; CSC: cancer stem cell; CTL: cytotoxic T lymphocyte; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FIGO: International Federation of Gynecology and Obstetrics; GFP: green fluorescent protein; GZMB: granzyme B; HG-CIN: high-grade CIN; IHC: immunohistochemistry; LG-CIN: low-grade CIN; LN: lymph node; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCL1: myeloid cell leukemia sequence 1; MLANA/MART-1: melanoma antigen recognized by T cells 1; MUT: mutant; NANOG: Nanog homeobox; PDCD1/PD-1: programmed cell death 1; PMEL/gp100: premelanosome protein; RTK: receptor tyrosine kinase; TMA: tissue microarray; WT: wild type.

HSP90A inhibition promotes anti-tumor immunity by reversing multi-modal resistance and stem-like property of immune-refractory tumors.

Cancer immunotherapy has emerged as a promising cancer treatment. However, the presence of immune-refractory tumor cells limits its clinical success by blocking amplification of anti-tumor immunity. Previously, we found that immune selection by immunotherapy drives the evolution of tumors toward multi-modal resistant and stem-like phenotypes via transcription induction of AKT co-activator TCL1A by NANOG. Here, we report a crucial role of HSP90A at the crossroads between NANOG-TCL1A axis and multi-aggressive properties of immune-edited tumor cells by identifying HSP90AA1 as a NANOG transcriptional target. Furthermore, we demonstrate that HSP90A potentiates AKT activation through TCL1A-stabilization, thereby contributing to the multi-aggressive properties in NANOGhigh tumor cells. Importantly, HSP90 inhibition sensitized immune-refractory tumor to adoptive T cell transfer as well as PD-1 blockade, and re-invigorated the immune cycle of tumor-reactive T cells. Our findings implicate that the HSP90A-TCL1A-AKT pathway ignited by NANOG is a central molecular axis and a potential target for immune-refractory tumor.

Inhibition of Carbonyl Reductase 1 Enhances Metastasis of Head and Neck Squamous Cell Carcinoma through ß-catenin-Mediated Epithelial-Mesenchymal Transition.

Objective: Human carbonyl reductase 1 (CBR1) plays key roles in the regulation of oxidative stress and tumor progression. However, the detailed mechanism and clinical correlation between CBR1 and tumor progression in head and neck squamous cell carcinoma (HNSCC) is largely unexplored. This study will focus the effects of CBR1 on head and neck cancer progression and explore the possible mechanisms. Materials and Methods: CBR1 mRNA expression was analyzed according to lymph node metastasis (LNM) status in patients with HNSCC from publicly available databases. CBR1 protein levels were measured and compared in HNSCC patient tissues, with or without metastasis, using immunohistochemistry (IHC). The invasive ability of HNSCC with modulated CBR1 expression was assayed using an invasion assay. Expression levels of EMT marker proteins were analyzed using immunoblotting. Results: HNSCC patients with LNM showed lower expression of CBR1 than those without LNM. In addition, IHC in tissues indicated that patients with LNM had relatively lower levels of CBR1 in cancer tissue. Consistently, in vitro invasion assay, we found that CBR1 inhibition using specific short interfering RNA treatment resulted in two- to three-fold increased invasion ability of HNSCC cell lines. Also, we proved that depletion of CBR1 activated marker proteins participating in epithelial-mesenchymal transition (EMT) signaling. CBR1 inhibition increased levels of intracellular reactive oxygen species (ROS) in HNSCC cells leading to upregulation of ß-catenin, one of main transcription factors that induce EMT-related genes. Conclusion: Our findings suggested that CBR1 plays an important role in metastasis of HNSCC tumors via regulation of ROS-mediated ß-catenin activity, and that CBR1 may be marker for progression of HNSCC to metastasis.

Mitochondrial reprogramming via ATP5H loss promotes multimodal cancer therapy resistance.

The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.

Stabilization of HDAC1 via TCL1-pAKT-CHFR axis is a key element for NANOG-mediated multi-resistance and stem-like phenotype in immune-edited tumor cells.

Cancer immunoediting enriches NANOG expression in tumor cells, resulting in multi-drug resistance and stem-like phenotypes. We previously demonstrated that these NANOG-associated phenotypes are promoted through HDAC1 transcriptional upregulation. In this study, we identified that NANOG also contributes to the stabilization of HDAC1 protein through the AKT signaling pathway. NANOG-AKT axis leads to phosphor-dependent inactivation of CHFR, an E3 ligase for HDAC1 protein, and thereby inhibiting the ubiquitin-mediated degradation of HDAC1. Furthermore, AKT inhibition disrupts HDAC1 WT-mediated phenotypes but had no effect on the phenotypes mediated by HDAC1 FM, a mutant that is unable to interact with CHFR. Critically, we applied a catalytic dead mutant, HDAC1-H141A, to uncover that HDAC1 confers immune-resistance, drug-resistance and stem-like phenotype in tumor cells through its catalytic activity. Collectively, our results establish a firm molecular link in immune-edited tumor cells among NANOG, AKT, CHFR, and HDAC1, identifying HDAC1 as a molecular target in controlling NANOGHIGH immune-refractory cancer.

Targeting Cyclin D-CDK4/6 Sensitizes Immune-Refractory Cancer by Blocking the SCP3-NANOG Axis.

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen-specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOGhigh cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3high immune-refractory cancer.Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3high immune-refractroy cancer. Cancer Res; 78(10); 2638-53. ©2018 AACR.

API5 induces cisplatin resistance through FGFR signaling in human cancer cells.

Most tumors frequently undergo initial treatment with a chemotherapeutic agent but ultimately develop resistance, which limits the success of chemotherapies. As cisplatin exerts a high therapeutic effect in a variety of cancer types, it is often used in diverse strategies, such as neoadjuvant, adjuvant and combination chemotherapies. However, cisplatin resistance has often manifested regardless of cancer type, and it represents an unmet clinical need. Since we found that API5 expression was positively correlated with chemotherapy resistance in several specimens from patients with cervical cancer, we decided to investigate whether API5 is involved in the development of resistance after chemotherapy and to explore whether targeting API5 or its downstream effectors can reverse chemo-resistance. For this purpose, cisplatin-resistant cells (CaSki P3 CR) were established using three rounds of in vivo selection with cisplatin in a xenografted mouse. In the CaSki P3 CR cells, we observed that API5 acted as a chemo-resistant factor by rendering cancer cells resistant to cisplatin-induced apoptosis. Mechanistic investigations revealed that API5 mediated chemo-resistance by activating FGFR1 signaling, which led to Bim degradation. Importantly, FGFR1 inhibition using either an siRNA or a specific inhibitor disrupted cisplatin resistance in various types of API5high cancer cells in an in vitro cell culture system as well as in an in vivo xenograft model. Thus, our results demonstrated that API5 promotes chemo-resistance and that targeting either API5 or its downstream FGFR1 effectors can sensitize chemo-refractory cancers.

HDAC1 Upregulation by NANOG Promotes Multidrug Resistance and a Stem-like Phenotype in Immune Edited Tumor Cells.

Cancer immunoediting drives the adaptation of tumor cells to host immune surveillance. Immunoediting driven by antigen (Ag)-specific T cells enriches NANOG expression in tumor cells, resulting in a stem-like phenotype and immune resistance. Here, we identify HDAC1 as a key mediator of the NANOG-associated phenotype. NANOG upregulated HDAC1 through promoter occupancy, thereby decreasing histone H3 acetylation on K14 and K27. NANOG-dependent, HDAC1-driven epigenetic silencing of cell-cycle inhibitors CDKN2D and CDKN1B induced stem-like features. Silencing of TRIM17 and NOXA induced immune and drug resistance in tumor cells by increasing antiapoptotic MCL1. Importantly, HDAC inhibition synergized with Ag-specific adoptive T-cell therapy to control immune refractory cancers. Our results reveal that NANOG influences the epigenetic state of tumor cells via HDAC1, and they encourage a rational application of epigenetic modulators and immunotherapy in treatment of NANOG+ refractory cancer types. Cancer Res; 77(18); 5039-53. ©2017 AACR.