FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity.

Nasopharyngeal carcinoma (NPC)-mediated immunosuppression within the tumor microenvironment (TME) frequently culminates in the failure of otherwise promising immunotherapies. In this study, we identify tumor-intrinsic FLI1 as a critical mediator in impairing T cell anti-tumor immunity. A mechanistic inquiry reveals that FLI1 orchestrates the expression of CBP and STAT1, facilitating chromatin accessibility and transcriptional activation of IDO1 in response to T cell-released IFN-γ. This regulatory cascade ultimately leads to augmented IDO1 expression, resulting in heightened synthesis of kynurenine (Kyn) in tumor cells. This, in turn, fosters CD8+ T cell exhaustion and regulatory T cell (Treg) differentiation. Intriguingly, we find that pharmacological inhibition of FLI1 effectively obstructs the CBP/STAT1-IDO1-Kyn axis, thereby invigorating both spontaneous and checkpoint therapy-induced immune responses, culminating in enhanced tumor eradication. In conclusion, our findings delineate FLI1-mediated Kyn metabolism as an immune evasion mechanism in NPC, furnishing valuable insights into potential therapeutic interventions.

Construction and validation of a macrophage polarization-related prognostic index to predict the overall survival in patients with early-stage triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and the current prognostic system cannot meet the clinical need. Interactions between immune responsiveness and tumor cells plays a key role in the progression of TNBC and macrophages are vital component of immune cells. A prognostic model based on macrophages may have great accuracy and clinical utility. Methods: For model development, we screened early stage (without metastasis) TNBC patients from The Cancer Genome Atlas (TCGA) database. We extracted messenger RNA (mRNA) expression data and clinical data including age, race, tumor size, lymph node status and tumor stage. The follow up time and vital status were also retrieved for overall survival calculation. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) was used to calculate the immune cell composition of each sample. Weighted gene co-expression network analysis (WGCNA) was used to identify M1-like macrophage-related genes. Combining least absolute shrinkage and selection operator (LASSO) with multivariate Cox regression, the M1-like macrophage polarization-related prognostic index (MRPI) was established. We obtained TNBC patients in Gene Expression Omnibus (GEO) database through PAM50 method and retrieved the mRNA expression data and survival data. The Harrell's concordance index (CI), the area under the receiver operating characteristic (ROC) curves (AUCs) and the calibration curve were used to evaluate the developed model. Results: We obtained 166 early TNBC cases and 113 normal tissue cases for model building, along with 76 samples from GSE58812 cohort for model validation. CIBERSORT analysis suggested obvious infiltration of macrophages, especially M1-like macrophages in early TNBC. Four genes were eventually identified for the construction of MPRI in the training set. The AUCs at 2 years, 3 years, and 5 years in the training cohort were 0.855, 0.881 and 0.893, respectively; and the AUCs at 2 years, 3 years, and 5 years in the validation cohort were 0.887, 0.792 and 0.722, respectively. Calibration curves indicated good predictive ability and high consistency of our model. Conclusions: MRPI is a promising biomarker for predicting the prognosis of early-stage TNBC, which may indicate personalized treatment and follow-up strategies and thus may improve the prognosis.

FLI1 regulates radiotherapy resistance in nasopharyngeal carcinoma through TIE1-mediated PI3K/AKT signaling pathway.

BACKGROUND: Radiotherapy resistance is the main cause of treatment failure in nasopharyngeal carcinoma (NPC), which leads to poor prognosis. It is urgent to elucidate the molecular mechanisms underlying radiotherapy resistance. METHODS: RNA-seq analysis was applied to five paired progressive disease (PD) and complete response (CR) NPC tissues. Loss-and gain-of-function assays were used for oncogenic function of FLI1 both in vitro and in vivo. RNA-seq analysis, ChIP assays and dual luciferase reporter assays were performed to explore the interaction between FLI1 and TIE1. Gene expression with clinical information from tissue microarray of NPC were analyzed for associations between FLI1/TIE1 expression and NPC prognosis. RESULTS: FLI1 is a potential radiosensitivity regulator which was dramatically overexpressed in the patients with PD to radiotherapy compared to those with CR. FLI1 induced radiotherapy resistance and enhanced the ability of DNA damage repair in vitro, and promoted radiotherapy resistance in vivo. Mechanistic investigations showed that FLI1 upregulated the transcription of TIE1 by binding to its promoter, thus activated the PI3K/AKT signaling pathway. A decrease in TIE1 expression restored radiosensitivity of NPC cells. Furthermore, NPC patients with high levels of FLI1 and TIE1 were correlated with poor prognosis. CONCLUSION: Our study has revealed that FLI1 regulates radiotherapy resistance of NPC through TIE1-mediated PI3K/AKT signaling pathway, suggesting that targeting the FLI1/TIE1 signaling pathway could be a potential therapeutic strategy to enhance the efficacy of radiotherapy in NPC.

Prognostic value of JAK3 promoter methylation and mRNA expression in clear cell renal cell carcinoma.

INTRODUCTION: Janus kinase 3 (JAK3) is a well-established oncogene in clear cell renal cell carcinoma (ccRCC). The methylation status of oncogene promoters has emerged as biomarkers for cancer diagnosis and prognosis. OBJECTIVE: This study aims to investigate the biological and clinical significance of JAK3 promoter methylation in ccRCC. METHODS: We analyzed the relationship of JAK3 promoter methylation with its mRNA expression, overall survival, and immune cell infiltration in a cohort obtained from The Cancer Genome Atlas (TCGA), which was further validated by another independent cohort. We further validated correlations of JAK3 promoter methylation with JAK3 expression, overall survival, and immune cell infiltration in an independent ccRCC cohort (Sun Yat-sen University Cancer Center (SYSUCC) cohort) by methods of immunohistochemistry (IHC) and pyrosequencing. RESULTS: We found JAK3 promoter was significantly hypomethylated in tumor tissues compared to normal adjacent tissues in ccRCC, and JAK3 promoter hypomethylation was strongly correlated with high JAK3 mRNA expression in all three ccRCC cohorts we examined. JAK3 promoter hypomethylation predicted advanced clinicopathological characteristics and shorter overall survival (TCGA cohort and SYSUCC cohort). Furthermore, we found that JAK3 promoter methylation was significantly associated with immune cell infiltration and expression of immune checkpoint molecules (TCGA cohort and CPTAC cohort). Finally, our SYSUCC cohort validated that JAK3 promoter methylation was correlated with CD4+ and CD8+ T cell infiltration in ccRCC tumor tissues. CONCLUSION: Our data demonstrated that the crucial role of JAK3 promoter methylation in its expression regulation and tumor microenvironment. JAK3 promoter methylation and expression are associated with clinicopathological characteristics, overall survival, and immune cell infiltration in ccRCC. We propose a rationale for further validation of JAK3 promoter methylation as a molecular biomarker for predicting responses to immune checkpoint inhibitors in ccRCC.

SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer.

Cancer cells modulate their metabolic activities to adapt to their growth and proliferation. Despite advances in breast cancer biology having led to the widespread use of molecular targeted therapy and hormonal drugs, the molecular mechanisms in metabolism related to the regulation of breast cancer cell proliferation are still poorly understood. Here, we investigate the possible role of SHMT2, a key enzyme in serine metabolism, in breast cancer. Firstly, SHMT2 is found highly expressed in both breast cancer cells and tissues, and patients with high expression of SHMT2 have a worse prognosis. Moreover, the intervention of SHMT2 by either knockdown or over-expression in vitro induces the effect on breast cancer proliferation. Mechanistically, RNA-seq shows that over-expression of SHMT2 affect multiple signaling pathways and biological process in breast cancer cells. Furthermore, we confirm that SHMT2 promotes breast cancer cell growth through MAPK and VEGF signaling pathways. Finally, we verify the role of SHMT2 in promoting breast cancer growth in the xenograft tumor model. Our results indicate that SHMT2 plays a critical role in regulating breast cancer growth through MAPK, and VEGF signaling pathways, and maybe serve as a therapeutic target for breast cancer therapy.

Nucleoporin TPR promotes tRNA nuclear export and protein synthesis in lung cancer cells.

The robust proliferation of cancer cells requires vastly elevated levels of protein synthesis, which relies on a steady supply of aminoacylated tRNAs. Delivery of tRNAs to the cytoplasm is a highly regulated process, but the machinery for tRNA nuclear export is not fully elucidated. In this study, using a live cell imaging strategy that visualizes nascent transcripts from a specific tRNA gene in yeast, we identified the nuclear basket proteins Mlp1 and Mlp2, two homologs of the human TPR protein, as regulators of tRNA export. TPR expression is significantly increased in lung cancer tissues and correlated with poor prognosis. Consistently, knockdown of TPR inhibits tRNA nuclear export, protein synthesis and cell growth in lung cancer cell lines. We further show that NXF1, a well-known mRNA nuclear export factor, associates with tRNAs and mediates their transport through nuclear pores. Collectively, our findings uncover a conserved mechanism that regulates nuclear export of tRNAs, which is a limiting step in protein synthesis in eukaryotes.

TNF Patterns and Tumor Microenvironment Characterization in Head and Neck Squamous Cell Carcinoma.

Background: HNSCC is a heterogeneous disease, which arises from distinct anatomic subsites, associates with various risk factors and possesses diverse molecular pathological features. Generally, HNSCC is considered as an immunosuppressive disease, characterized by abnormal tumor immune microenvironment. The TNF family plays a crucial role in the survival, proliferation, differentiation, and effector functions in both immune and non-immune cells. However, the expression patterns of TNF in HNSCC remains to be systematically analyzed. Methods: We downloaded transcriptional profile data of HNSCC from TCGA and GEO datasets. Unsupervised clustering methods were used to identify different TNF patterns and classify patients for further analysis. PCA was conducted to construct a TNF relevant score, which we called risk score. Results: In this study, we systematically evaluated the patterns of TNF family and tumor immune microenvironment characteristics of HNSCC patients by clustering the expression of 46 members of TNF family. We identified two subtypes with distinct clinical and immune characteristics in HNSCC and constructed a risk scoring system based on the expression profile of TNF family genes. Conclusion: Risk score serves as a reliable predictor of overall survival, clinical characteristics, and immune cell infiltration, which has the potential to be applied as a valuable biomarker for HNSCC immunotherapy.

Fermitin family member 2 promotes melanoma progression by enhancing the binding of p-α-Pix to Rac1 to activate the MAPK pathway.

We identified fermitin family member 2 (FERMT2, also known as kindlin-2) as a potential target in A375 cell line by siRNA library screening. Drugs that target mutant BRAF kinase lack durable efficacy in the treatment of melanoma because of acquired resistance, thus the identification of novel therapeutic targets is needed. Immunohistochemistry was used to identify kindlin-2 expression in melanoma samples. The interaction between kindlin-2 and Rac1 or p-Rac/Cdc42 guanine nucleotide exchange factor 6 (α-Pix) was investigated. Finally, the tumor suppressive role of kindlin-2 was validated in vitro and in vivo. Analysis of clinical samples and Oncomine data showed that higher levels of kindlin-2 predicted a more advanced T stage and M stage and facilitated metastasis and recurrence. Kindlin-2 knockdown significantly inhibited melanoma growth and migration, whereas kindlin-2 overexpression had the inverse effects. Further study showed that kindlin-2 could specifically bind to p-α-Pix(S13) and Rac1 to induce a switch from the inactive Rac1-GDP conformation to the active Rac1-GTP conformation and then stimulate the downstream MAPK pathway. Moreover, we revealed that a Rac1 inhibitor suppressed melanoma growth and metastasis and the combination of the Rac1 inhibitor and vemurafenib resulted in a better therapeutic outcome than monotherapy in melanoma with high kindlin-2 expression and BRAF mutation. Our results demonstrated that kindlin-2 promoted melanoma progression, which was attributed to specific binding to p-α-Pix(S13) and Rac1 to stimulate the downstream MAPK pathway. Thus, kindlin-2 could be a potential therapeutic target for treating melanoma.

Poly(U) binding splicing factor 60 promotes renal cell carcinoma growth by transcriptionally upregulating telomerase reverse transcriptase.

Background: Abnormal transcriptional upregulation of telomerase reverse transcriptase (TERT) plays a dominant role in telomerase activation in various cancers. TERT promoter mutations (TPMs) have been identified as a key mechanism in TERT upregulation. However, the mechanism of TERT upregulation in cancers with low frequency of TPMs are not fully elucidated so far. Methods: The expression of PUF60 and TERT was detected by real-time PCR, western blot and immunohistochemistry. TERT promoter binding proteins were identified by streptavidin-agarose pulldown assay and mass spectrum (MS) analysis. The role of PUF60/TERT in renal cancer was evaluated on cell growth in vitro and in vivo. Results: In this study, we identify the regulation mechanism of TERT in renal cell carcinoma (RCC) cells which have rare TPMs but exert significant upregulation of TERT. We found that TERT was highly expressed in RCC tumor tissues, and elevated TERT expression was associated with poor prognosis for patients. We also detected the relatively rare TPM status in both RCC tumor tissues and RCC cell lines. Mechanistically, PUF60, a RNA binding protein, was identified as a novel TERT regulator which bound to the TERT and transcriptionally upregulated TERT expression in RCC cells. The in vitro and in vivo experiments also demonstrated that PUF60 could promote RCC cell growth through activation of TERT expression in a TPM status independent way. Furthermore, we showed that there was a strong correlation of the expression of PUF60 and TERT in RCC tumor tissues and RCC cell lines, and the patients with high expression of PUF60 and TERT had significantly shorter survival. Conclusions: Collectively, these results indicated that PUF60 transcriptionally upregulated TERT expression to promote RCC growth and progression in a TPM status independent way, suggesting that the PUF60/TERT signaling pathway may serve as potential prognostic biomarkers and therapeutic targets for RCC.

KMT2A regulates cervical cancer cell growth through targeting VDAC1.

Cervical cancer is an aggressive cutaneous malignancy, illuminating the molecular mechanisms of tumorigenesis and discovering novel therapeutic targets are urgently needed. KMT2A is a transcriptional co-activator regulating gene expression during early development and hematopoiesis, but the role of KMT2A in cervical cancer remains unknown. Here, we demonstrated that KMT2A regulated cervical cancer growth via targeting VADC1. Knockdown of KMT2A significantly suppressed cell proliferation and migration and induced apoptosis in cervical cancer cells, accompanying with activation of PARP/caspase pathway and inhibition of VADC1. Overexpression of VDAC1 reversed the KMT2A knockdown-mediated regulation of cell proliferation, migration and apoptosis. The in vivo results from a cervical cancer xenograft mouse model also validated that KMT2A knockdown suppressed tumor growth by inhibiting VDAC1, whereas KMT2A overexpression promoted cervical cancer growth. Moreover, analyses of Biewenga cervix database and clinical samples showed that both KMT2A and VDAC1 were upregulated in cervix squamous cell carcinoma compared with cervix uteri tissues, and their expression was negatively correlated with the differentiation grade of cervical cancer. Our results therefore indicated that the KMT2A/VDAC1 signaling axis may be a potential new mechanism of cervical carcinogenesis.

NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer.

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.

High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma.

According to our previous study, GOLPH3 is markedly up-expressed in tongue squamous cell carcinoma (TSCC), which is also associated with poor survival. However, it remains unclear about key upstream and downstream mechanisms of GOLPH3. This study aimed to illuminate new mechanisms modulating GOLPH3 upregulation and promoting TSCC development at the molecular level. Using mass spectrometry and agarose-streptavidin-biotin pull-down analyses, SOX8 (SRY-Box 8) was identified to be the new protein to bind the GOLPH3 promoter within TSCC cells, which was further verified to be the regulator of GOLPH3 upregulation. The knockdown of SOX8 suppressed the promoter activity of GOLPH3, while secondarily inhibiting TSCC cell proliferation both in vivo and in vitro. Interestingly, GOLPH3 overexpression rescued the SOX8 knockdown-mediated suppression on TSCC proliferation. Additionally, exogenous over-expression of SOX8 also activated the activity of promoter as well as GOLPH3 expression, in the meantime of promoting TSCC development. Moreover it was discovered that SOX8 regulated GOLPH3 expression through interacting with TFAP2A. Moreover our results suggested that the SOX8 level was increased within tumor tissue compared with that in para-cancer normal counterpart, which showed positive correlation with the GOLPH3 level. According to Kaplan-Meier analyses, TSCC cases having higher SOX8 and GOLPH3 expression were associated with poorer prognostic outcomes. Taken together, this study reveals that SOX8 enhances the TSCC cell growth via the direct transcriptional activation of GOLPH3, which also indicates the potential to use SOX8/GOLPH3 pathway as the treatment target among TSCC patients.

Expression of PD-1/PD-L1 in head and neck squamous cell carcinoma and its clinical significance.

OBJECTIVE: To investigate the role of programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and P16 in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 95 paraffin-embedded samples of tumorous tissue of HNSCC were collected. Expression levels of PD-1, PD-L1, and P16 were determined by immunohistochemistry. RESULTS: A significantly higher proportion of PD-1 among patients infected with the human papillomavirus was found. PD-L1 expression is closely associated with the primary site of the tumor, postoperative recurrence, survival, PD-1 expression and P16 expression. Univariable analysis indicated that T stage, N stage, tumor node metastasis stage, tumor differentiation, and PD-L1 expression were all shown to be prognostic variables for overall survival in patients with HNSCC. In the multivariate analysis, only N stage (P = 0.010) and PD-L1 expression (P = 0.001) were found to be independent prognostic variables for overall survival. In addition, for disease recurrence, multivariate analysis showed that only PD-L1 expression was the associated independent risk factor. For the patients with negative PD-L1 expression, Kaplan-Meier analysis revealed that they had significantly worse outcomes in terms of overall survival (P = 0.001). Similarly, compared with the patients with positive PD-L1 expression, those with negative PD-L1 expression had a higher probability of recurrence (P = 0.026). CONCLUSIONS: The expression of PD-L1, PD-1, and P16 in HNSCC is significantly correlated. Human papillomavirus infection (P16 positive) is negatively related to postoperative recurrence. HNSCC patients with positive PD-L1/PD-1 expression tend to have better overall survival outcomes and lower probability of recurrence, providing more evidence for the PD-l-targeted immunotherapy of HNSCC.

Melatonin enhances sorafenib-induced cytotoxicity in FLT3-ITD acute myeloid leukemia cells by redox modification.

Acute myeloid leukemia (AML) with an internal tandem duplication in Fms-related tyrosine kinase 3 (FLT3-ITD) is identified as a subgroup with poor outcome and intrinsic resistance to chemotherapy and therefore urgent need for development of novel therapeutic strategies. Methods: The antitumor effects of melatonin alone or combined with sorafenib were evaluated via flow cytometry and immunoblotting assays in FLT-ITD AML cells. Also, the ex vivo and in vivo models were used to test the synergistic effects of melatonin and sorafenib against leukemia with FLT3/ITD mutation. Results: Our study shows for the first time that melatonin inhibits proliferation and induces apoptosis in FLT3/ITD-positive leukemia cells. Mechanistically, melatonin preferentially causes overproduction of reactive oxygen species (ROS) and ultimately massive cell death in FLT3-ITD AML cells. Moreover, melatonin significantly enhances the cytotoxicity induced by the FLT3 tyrosine kinase inhibitor sorafenib in AML cells with FLT3/ITD through redox modification. Importantly, combination of melatonin and sorafenib exhibited highly synergistic therapeutic activity in MV4-11 xenografts and a murine model bearing FLT3/ITD leukemia. Conclusion: This study indicates that melatonin, alone or in combination with sorafenib, has potential to improve the therapeutic outcome of AML patients with FLT3-ITD mutation that merits further investigation.

Metformin mediates induction of miR-708 to inhibit self-renewal and chemoresistance of breast cancer stem cells through targeting CD47.

Breast cancer stem cells (BCSCs) have been considered responsible for cancer progression, recurrence, metastasis and drug resistance. However, the mechanisms by which cells acquire self-renewal and chemoresistance properties are remaining largely unclear. Herein, we evaluated the role of miR-708 and metformin in BCSCs, and found that the expression of miR-708 is significantly down-regulated in BCSCs and tumour tissues, and correlates with chemotherapy response and prognosis. Moreover, miR-708 markedly inhibits sphere formation, CD44+ /CD24- ratio, and tumour initiation and increases chemosensitivity of BCSCs. Mechanistically, miR-708 directly binds to cluster of differentiation 47 (CD47), and regulates tumour-associated macrophage-mediated phagocytosis. On the other hand, CD47 is essential for self-renewal, tumour initiation and chemoresistance of BCSCs, and correlates with the prognosis of breast cancer patients. In addition, the anti-type II diabetes drug metformin are found to be involved in the miR-708/CD47 signalling pathway. Therefore, our study demonstrated that miR-708 plays an important tumour suppressor role in BCSCs self-renewal and chemoresistance, and the miR-708/CD47 regulatory axis may represent a novel therapeutic mechanism of metformin in BCSCs.

TFAP2B overexpression contributes to tumor growth and progression of thyroid cancer through the COX-2 signaling pathway.

Thyroid cancer is commonly seen in the clinic with a rapidly increasing incidence globally. COX-2 overexpression correlates with the pathologic type of thyroid carcinoma, and it has been suggested that COX-2 overexpression is associated with a poor prognosis. However, little is known about its upstream regulatory mechanism. Bioinformatics suggested that transcription factor AP-2 beta (TFAP2B) might specifically bind to the COX-2 promoter, which was confirmed by biotin-labeled COX-2 promoter pulldown and luciferase reporter assays. We performed western blot and immunohistochemical staining to detect the expression of TFAP2B/COX-2 in thyroid cancer tissues (T) and the matched adjacent noncarcinoma tissues (ANT), and investigated the relationship between TFAP2B/COX-2 expression and clinical pathological factors in thyroid cancer patients. Afterward, MTS, colony formation, cell-apoptosis assay, transwell-invasion and scratch assays were performed to examine the proliferation, apoptosis, invasion, and migration of thyroid cancer cells with TFAP2B knocked down or overexpressed. The mouse xenograft experiment was performed to study in vivo the proliferation of thyroid cancer cells with TFAP2B knocked down or overexpressed. We found that TFAP2B bound to the promoter of COX-2 to activate its expression. Western blot and immunohistochemistry showed that TFAP2B/COX-2 was highly expressed in thyroid cancer, and high TFAP2B and COX-2 expression was associated with aggressive clinicopathological features in thyroid cancer. TFAP2B mediated thyroid cancer cell proliferation, apoptosis, invasion, and migration via the COX-2 signaling pathway in vitro and in vivo. TFAP2B bound to the promoter of COX-2 to activate its expression, indicating that TFAP2B is a critical regulatory molecule in the COX-2 signaling pathway that promoted tumor progression in thyroid cancer.

Panobinostat (LBH589) inhibits Wnt/ß-catenin signaling pathway via upregulating APCL expression in breast cancer.

Breast cancer is the most common malignant disease among women worldwide and the novel therapeutic agents are urgently needed. Panobinostat (LBH589), a pan-HDACs inhibitor, has shown promising anti-tumor effect in recent years. However, the targets of this compound are largely unclear because of its low selectivity. In consideration of the transcription promoting activity of panobinostat, we speculated that specific tumor suppressor genes might be upregulated after panobinostat treatment. In this study, we verified the inhibition effect of panobinostat in different subtypes of breast cancer cells in vivo and in vitro. We found that panobinostat suppressed proliferation, migration as well as invasion, and induced apoptosis in both TNBC and non-TNBC cells. Consistently, panobinostat inhibited breast cancer growth and metastasis in mouse models. Mechanistically, we found APCL transcription and expression was significantly upregulated in panobinostat treated cells by RNA microarray analysis, while knockdown of APCL resulted in reduced sensitivity to panobinostat in breast cancer cells. APCL is a wnt/ß-catenin pathway regulator that promotes ß-catenin ubiquitylation and degradation. We found that panobinostat inhibited ß-catenin expression by increasing its ubiquitylation and thus reducing its half-life. In addition, the expression of ß-catenin activated targets including c-Jun, c-Myc, Cyclin D1 and CD44 were also decreased by panobinostat treatment in breast cancer cells. These results suggested that panobinostat inhibited tumor growth and metastasis via upregulating APCL expression in breast cancer cells, which was a novel and crucial mechanism of panobinostat.

Identification of a 4-mRNA metastasis-related prognostic signature for patients with breast cancer.

Metastasis-related mRNAs have showed great promise as prognostic biomarkers in various types of cancers. Therefore, we attempted to develop a metastasis-associated gene signature to enhance prognostic prediction of breast cancer (BC) based on gene expression profiling. We firstly screened and identified 56 differentially expressed mRNAs by analysing BC tumour tissues with and without metastasis in the discovery cohort (GSE102484, n = 683). We then found 26 of these differentially expressed genes were associated with metastasis-free survival (MFS) in the training set (GSE20685, n = 319). A metastasis-associated gene signature built using a LASSO Cox regression model, which consisted of four mRNAs, can classify patients into high- and low-risk groups in the training cohort. Patients with high-risk scores in the training cohort had shorter MFS (hazard ratio [HR] 3.89, 95% CI 2.53-5.98; P < 0.001), disease-free survival (DFS) (HR 4.69, 2.93-7.50; P < 0.001) and overall survival (HR 4.06, 2.56-6.45; P < 0.001) than patients with low-risk scores. The prognostic accuracy of mRNAs signature was validated in the two independent validation cohorts (GSE21653, n = 248; GSE31448, n = 246). We then developed a nomogram based on the mRNAs signature and clinical-related risk factors (T stage and N stage) that predicted an individual's risk of disease, which can be assessed by calibration curves. Our study demonstrated that this 4-mRNA signature might be a reliable and useful prognostic tool for DFS evaluation and will facilitate tailored therapy for BC patients at different risk of disease.

MAD2L2 inhibits colorectal cancer growth by promoting NCOA3 ubiquitination and degradation.

Nuclear receptor coactivator 3 (NCOA3) is a transcriptional coactivator that has elevated expression in multiple tumor types, including colorectal cancer (CRC). However, the molecular mechanisms that regulate the tumorigenic functions of NCOA3 in CRC remain largely unknown. In this study, we aimed to discover and identify the novel regulatory proteins of NCOA3 and explore their mechanisms of action. Immunoprecipitation (IP) coupled with mass spectrometry (IP-MS) analysis was used to detect, identify, and verify the proteins that interacted with NCOA3 in CRC cells. The biological functions of the candidate proteins and the underlying molecular mechanism were investigated in CRC cells and mouse model in vitro and in vivo. The clinical significance of NCOA3 and its interaction partner protein in CRC patients was also studied. We identified mitotic arrest deficient 2-like protein 2 (MAD2L2, also known as MAD2B or REV7), with two signal peptide sequences of LIPLK and EVYPVGIFQK, to be an interaction partner of NCOA3. Overexpression of MAD2L2 suppressed the proliferation, migration, and clonogenicity of CRC cells by inducing the degradation of NCOA3. The mechanism study showed that increased MAD2L2 expression in CRC cells activated p38, which was required for the phosphorylation of NCOA3 that led to its ubiquitination and degradation by the proteasome. Moreover, we found that MAD2L2 predicted favorable prognosis in CRC patients. We have discovered a novel role of MAD2L2 in the regulation of NCOA3 degradation and proposed that MAD2L2 serves as a tumor suppressor in CRC.

PD-1 and PD-L1 expression in 132 recurrent nasopharyngeal carcinoma: the correlation with anemia and outcomes.

The expression of Programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) has been reported to be reliable prognostic factors in various malignances including primary nasopharyngeal carcinoma (NPC). However, the exact role of PD-1/PD-L1 in recurrent NPC remains unclear. In this study, we aimed to investigate the relationship between the expression of PD-1 / PD-L1 and the clinical-pathology as well the outcomes of recurrent NPC patients (n = 132). The expression of PD-1 and PD-L1 was measured by immunohistochemistry staining. The relationship between PD-1 / PD-L1 and factors involved in clinic-pathology and outcomes of patients with NPC was assessed by correlation analysis. To further explore the association between PD-L1 and anemia, immunofluorescence analysis was performed to investigate the correlation of PD-L1 with hypoxia inducible factor-1α (HIF-1α). We observed that advanced rT classification and anemia status before salvage treatment was associated with high level of PD-L1 in recurrent NPC patients, and PD-L1 and was co-located with HIF-1α in recurrent tumors by immunofluorescence analysis. Moreover, our result suggested that PD-L1 might be a negative indicator for recurrent NPC patients as well as age, rT classification, anemia and tumor necrosis at diagnose of recurrence. Taken together, our results revealed that PD-L1 might be a potential prognostic biomarker for recurrent NPC patients, and advanced re-stage, anemia might represent as candidate biomarkers for evaluating patients' response to anti-PD-1 / PD-L1-treatment. However, further studies are needed to clarify the underlying mechanism of hypoxia in immunosuppression process induced by PD-1 / PD-L1 axis.