ABSTRACT
BACKGROUND: Forkhead box F2 (FOXF2) is relatively limited to the adult lung, but its contribution to non-small cell lung cancer (NSCLC) prognosis is unclear. RESULTS: FOXF2 mRNA levels in NSCLC were lower than that in paired normal lung tissues (P = 0.012). The FOXF2low patients had shorter survival time than the FOXF2high patients (P = 0.024) especially in stage I (P = 0.002), chemotherapy (P = 0.018) and < 60 age groups (P = 0.002). Lower FOXF2 mRNA levels could independently predict poorer survival for patients with NSCLC (HR = 2.384, 95% CI = 1.241-4.577; P = 0.009), especially in stage I (HR =4.367, 95% CI =1.599-11.925; P = 0.004). The two independent datasets confirmed our findings. METHODS: We examined FOXF2 mRNA levels in 84 primary NSCLC and 8 normal lung tissues using qRT-PCR. Rank-sum tests and chi-square tests were used to assess the differences among groups with various clinicopathological factors. Kaplan-Meier tests were used to compare survival status in patients with different FOXF2 mRNA levels. Cox proportional hazards regression model was used to evaluate the predictive value of FOXF2 mRNA level in NSCLC patients. Independent validation was performed using an independent dataset (98 samples) and an online survival analysis software Kaplan-Meier plotter (1928 samples). CONCLUSIONS: Our results demonstrated that decreased FOXF2 expression is an independent predictive factor for poor prognosis of patients with NSCLC, especially in stage I NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/mortality , Forkhead Transcription Factors/analysis , Lung Neoplasms/mortality , Adult , Aged , Carcinoma, Non-Small-Cell Lung/chemistry , Carcinoma, Non-Small-Cell Lung/pathology , Female , Forkhead Transcription Factors/genetics , Humans , Lung Neoplasms/chemistry , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasm Staging , Prognosis , Proportional Hazards Models , RNA, Messenger/analysisABSTRACT
Forkhead box (FOX) F2 and FOXC2 belong to the FOX transcription factor superfamily. FOXC2 is recognized as an inducer of epithelial-mesenchymal transition (EMT), and its overexpression promotes basal-like breast cancer (BLBC) metastasis. Our previous study demonstrated that FOXF2 functions as an EMT suppressor and that FOXF2 deficiency promotes BLBC metastasis. However, the relationship between the opposite EMT-related transcription factors FOXF2 and FOXC2 remains unknown. Here, we found that FOXF2 directly targets FOXC2 to negatively regulate FOXC2 transcription in BLBC cells. Functionally, we observed that FOXC2 mediates the FOXF2-regulated EMT phenotype, aggressive behavior, and multiple chemotherapy drug resistance of BLBC cells. Additionally, we detected a significant negative correlation between the FOXF2 and FOXC2 mRNA levels in triple-negative breast cancer (TNBC) tissues. TNBC patients in the FOXF2high/FOXC2low and FOXF2low/FOXC2high groups exhibited the best and worst disease-free survival (DFS), respectively, whereas the patients in the FOXF2high/FOXC2high and FOXF2low/FOXC2low groups exhibited moderate DFS. In summary, we found that FOXF2 transcriptionally targets FOXC2 and suppresses EMT and multidrug resistance by negatively regulating the transcription of FOXC2 in BLBC cells. The combined expression levels of FOXF2 and FOXC2 mRNA might serve as an effective prognostic indicator and could guide tailored therapy for TNBC or BLBC patients.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition/drug effects , Forkhead Transcription Factors/metabolism , Signal Transduction/drug effects , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Disease-Free Survival , Dose-Response Relationship, Drug , Female , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , MCF-7 Cells , Phenotype , RNA Interference , RNA, Messenger/metabolism , Time Factors , Transcription, Genetic , Transfection , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
FOXF2 (forkhead box F2) is a mesenchyme-specific transcription factor that plays a critical role in tissue homeostasis through the maintenance of epithelial polarity. In a previous study, we demonstrated that FOXF2 is specifically expressed in basal-like breast cancer (BLBC) cells and functions as an epithelial-mesenchymal transition suppressor. FOXF2 deficiency enhances the metastatic ability of BLBC cells through activation of the epithelial-mesenchymal transition program, but reduces cell proliferation. In this study, we demonstrate that CpG island methylation of the FOXF2 proximal promoter region is involved in the regulatory mechanism of the subtype-specific expression of FOXF2 in breast cancer cells. DNMT1, DNMT3A, and DNMT3B commonly or individually contributed to this DNA methylation in different breast cancer cells. SP1 regulated the transcriptional activity of FOXF2 through direct binding to the proximal promoter region, whereas this binding was abrogated through DNA methylation. FOXF2 mediated the SP1-regulated suppression of progression and promotion of proliferation of non-methylated BLBC cells. Thus, we conclude that the subtype-specific expression and function of FOXF2 in breast cancer cells are regulated through the combined effects of DNA methylation and SP1 transcriptional regulation.
Subject(s)
Breast Neoplasms/genetics , DNA Methylation , Forkhead Transcription Factors/metabolism , Neoplasms, Basal Cell/genetics , Sp1 Transcription Factor/physiology , Base Sequence , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Cell Movement , Cell Proliferation , CpG Islands , Disease-Free Survival , Epigenesis, Genetic , Female , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , MCF-7 Cells , Molecular Sequence Data , Neoplasms, Basal Cell/metabolism , Neoplasms, Basal Cell/mortality , Promoter Regions, Genetic , Up-RegulationABSTRACT
Bone metastasis affects more than 70% of advanced breast cancer patients, but the molecular mechanisms of this process remain unclear. Here, we present clinical and experimental evidence to clarify the role of the integrin ß-like 1 (ITGBL1) as a key contributor to bone metastasis of breast cancer. In an in vivo model system and in vitro experiments, ITGBL1 expression promoted formation of osteomimetic breast cancers, facilitating recruitment, residence, and growth of cancer cells in bone microenvironment along with osteoclast maturation there to form osteolytic lesions. Mechanistic investigations identified the TGFß signaling pathway as a downstream effector of ITGBL1 and the transcription factor Runx2 as an upstream activator of ITGBL1 expression. In support of these findings, we also found that ITGBL1 was an essential mediator of Runx2-induced bone metastasis of breast cancer. Overall, our results illuminate how bone metastasis occurs in breast cancer, and they provide functional evidence for new candidate biomarkers and therapeutic targets to identify risk, to prevent, and to treat this dismal feature of advanced breast cancer.
Subject(s)
Bone Neoplasms/genetics , Breast Neoplasms/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Integrin beta1/genetics , Signal Transduction/genetics , Transforming Growth Factor beta/genetics , Animals , Blotting, Western , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/genetics , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Integrin beta1/metabolism , Kaplan-Meier Estimate , Mice, Inbred BALB C , Mice, SCID , Osteoblasts/metabolism , Osteogenesis/genetics , Osteolysis/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/metabolismABSTRACT
INTRODUCTION: Our previous clinical study demonstrated that the under-expression of FOXF2 is associated with early-onset metastasis and poor prognosis of patients with triple-negative breast cancer. In this study, we further characterized the role of FOXF2 in metastasis of basal-like breast cancer (BLBC) and underlying molecular mechanisms. METHODS: RT-qPCR, immunoblot, immunofluorescence and immunohistochemistry were performed to assess the expression of genes and proteins in cell lines and tissues. A series of in vitro and in vivo assays was performed in the cells with RNAi-mediated knockdown or overexpression to elucidate the function and transcriptional regulatory role of FOXF2 in breast cancer. RESULTS: We found that FOXF2 was specifically expressed in most basal-like breast cells. FOXF2 deficiency enhanced the metastatic ability of BLBC cells in vitro and in vivo. Additionally, FOXF2 deficiency induced the epithelial-mesenchymal transition (EMT) of basal-like breast cells. Furthermore, we identified that TWIST1 is a transcriptional target of FOXF2. TWIST1 was negatively regulated by FOXF2 and mediated the FOXF2-regulated EMT phenotype of basal-like breast cells and aggressive property of BLBC. CONCLUSIONS: FOXF2 is a novel EMT-suppressing transcription factor in BLBC. FOXF2 deficiency enhances metastatic ability of BLBC cells by activating the EMT program through upregulating the transcription of TWIST1.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/pathology , Epithelial-Mesenchymal Transition/genetics , Forkhead Transcription Factors/deficiency , Animals , Breast Neoplasms/metabolism , Carcinoma, Basal Cell/metabolism , Cell Line, Tumor , Cell Movement/genetics , Disease Models, Animal , Epithelial Cells/metabolism , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Mice , Neoplasm Metastasis , Nuclear Proteins/genetics , Phenotype , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Twist-Related Protein 1/geneticsABSTRACT
The glutathione-S-transferase (GST) family contributes to the inactivation of various toxic compounds formed as secondary metabolites during oxidative stress. GSTP1 accounts for the majority of the GST family enzymatic activity, and the activity of GSTP1 enzyme can be altered by the presence of the Ile105Val polymorphism. In this study, we examined the polymorphic frequency of GSTP1 Ile105Val genotype in 920 breast cancer patients and 783 healthy controls in women of North China. Results showed that GSTP1 105Val allele (Ile/Val and Val/Val) was associated with a higher breast cancer risk (OR = 1.38, 95% CI: 1.14-1.69; P = 0.001) and more aggressive tumors with histological grade III (OR = 1.15, 95% CI: 1.05-1.26; P = 0.001), lymph node metastases (OR = 2.35, 95% CI: 1.72-3.21; P < 0.001), as well as ER negative (OR = 1.77, 95% CI: 1.31-2.39; P < 0.001) than those carrying the Ile/Ile allele. However, the patients with the GSTP1 105Val genotype had a better disease free survival after cyclophosphamide (CTX)-based chemotherapy than those with Ile/Ile (HR = 0.77, 95% CI: 0.45-0.91; P < 0.001). Furthermore, in vitro cellular experiments demonstrated that breast cancer cells with the GSTP1 105Val allele were significantly more sensitive to CTX-induced proliferation inhibition. Thus, we conclude that the GSTP1 105Val allele increases breast cancer risk and aggressiveness and enhance response to CTX-based chemotherapy in women of North China. Detection of the GSTP1 Ile105Val genotype may help screen for high-risk populations and direct individualized therapy.
Subject(s)
Alleles , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Genetic Predisposition to Disease , Glutathione S-Transferase pi/genetics , Adult , Aged , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Case-Control Studies , China , Cyclophosphamide/therapeutic use , Disease-Free Survival , Female , Gene Frequency/genetics , Humans , Kaplan-Meier Estimate , Middle Aged , Multivariate Analysis , Neoplasm Invasiveness , Polymorphism, Single Nucleotide/genetics , Risk Factors , Young AdultABSTRACT
The transcription factor, FOXF2, plays an important role in tissue development, extracellular matrix synthesis, and epithelial-mesenchymal interactions, implying that it may be associated with the metastatic capabilities of cancer cells. However, the relationship between FOXF2 expression and breast cancer progression, metastasis, and prognosis, remains to be elucidated. In this study, FOXF2 mRNA levels in 305 primary breast cancer tissues were examined using RT-QPCR. Results showed that FOXF2 mRNA levels in primary breast cancer were negatively associated with tumor progression, including tumor size, number of metastatic lymph nodes, and clinical stage. Patients with low FOXF2 mRNA levels had a high risk of relapse and metastasis within three years. Low FOXF2 mRNA levels could predict shorter disease-free survival for those patients with histological grade II and triple-negative breast cancer. Taken together, we conclude that decreased FOXF2 expression indicates the early-onset metastasis and poor prognosis for patients with histological grade II and triple-negative breast cancer.
