FBI-1 inhibits epithelial-to-mesenchymal transition, migration, and invasion in lung adenocarcinoma A549 cells by downregulating transforming growth factor-ß1 signaling pathway.

The factor binding inducer of short transcripts-1 (FBI-1) is a POZ-domain Kruppel-like (POK) family of transcription factors and is known as a proto-oncogene or tumor suppressor in various carcinomas. However, the role of FBI-1 on epithelial-to-mesenchymal transition (EMT) and invasiveness in lung cancer remains unknown. Preliminarily, clinical data such as tissue microarray, Kaplan-Meier, and Oncomine were analyzed to confirm the correlation between lung cancer metastasis and FBI-1. To investigate the function of FBI-1 in EMT in lung cancer, EMT was measured in FBI-1-deficient or FBI-1-overexpressing cells. FBI-1 showed decreased expression in tumors metastasized to lymph nodes than in the primary tumor. In addition, it was also associated with improved survival rates of lung cancer patients. FBI-1 knockdown improved E-to-N-cadherin switching, migration, and invasion in A549 cells, similar to the initiation of EMT stimulated by transforming growth factor- ß1 (TGF-ß1). In contrast, overexpression of FBI-1 inhibited the transcription and activation of Smad2, thereby interfering with EMT, despite stimulation by TGF-ß1. These results suggest that FBI-1 plays a negative role in EMT in lung cancer via the TGF-ß1 signaling pathway, implying its use as a new potential therapeutic target and diagnostic indicator for early stage of lung cancer metastasis.

[Esculin inhibits proliferation of triple negative breast cancer cells by down-regulating FBI-1].

Objective: To investigate the effect of esculin on the proliferation of triple negative breast cancer cells and its molecular mechanism. Methods: MDA-MB-231 cells were treated with 28, 56, 112, 225, 450 and 900 µmol/L of esculin for 24, 48 and 72 h, respectively, and the cell viability was detected by cell counting kit 8 (CCK-8) assay. In addition, MDA-MB-231 cells were treated with 0, 225, 450 and 900 µmol/L of esculin for 48 h. And then the changes in cell morphology were observed by inverted microscope. The clone-forming ability was detected by colony formation assay. The mRNA expression levels of FBI-1, p53 and p21 were detected using real-time fluorescence quantitative polymerase chain reaction. The protein expression levels of FBI-1, p53, p21 and Ki67 were detected by western blot. Results: Compared with the blank control group, the cell viability of MDA-MB-231 cells that treated with esculin significantly decreased in a dose-dependent and time-dependent manners. After treatment with esculin, MDA-MB-231 cells shrunk, flattened, adhered poorly to the culture dish and the cell spacing became larger. Meanwhile, shedding and incomplete cells appeared, of which 900 µmol/L of esculin treatment group showed the most dramatic changes. In addition, the colony formation ratios were decreased to (77.18±5.13)%, (65.94±4.98)% and (45.92±3.70)% in the 225, 450 and 900 µmol/L of esculin treatment groups compared with blank control, respectively (P<0.01). Furthermore, the mRNA and protein expressions of FBI-1 increased, while the levels of p53 and p21 mRNA and protein, as well as the protein expression of Ki67 decreased in a concentration-dependent manner (P<0.01). Conclusion: Esculin may regulate cell cycle-related p53-p21 pathway via FBI-1 mediated DNA replication, thus inhibit the proliferation of triple negative breast cancer cells.

[Analyses of the Expression of FBI-1 in Breast Cancer Pre- and Pro-neoadjuvant Chemotherapy].

Objective: To investigate the expression of factor that binds to inducer of short transcripts-1 of HIV (FBI-1)in breast cancer pre- and pro-neoadjuvant chemotherapy and explore the relationship between FBI-1 expression and treatment efficacy. Methods: We collected 50 patients with breast cancer who received neoadjuvant chemotherapy before operation in the Affiliated Tumor Hospital of Guangxi Medical University from January, 2010 to December, 2014. The expression of FBI-1 in breast cancer tissues pre- and pro-neoadjuvant chemotherapy was detected by immunohistochemical staining. We compared the level of FBI-1 expression pre- and pro-neoadjuvant chemotherapy, and tried to explore its relationship with patient and tumor characteristics and treatment efficacy. Results: (1) The rate of upregulated expression of FBI-1 in breast cancer tissues was 70% (35/50). The upregulated expression of FBI-1 was related to the higher clinical stage and trend of lymph node metastasis (P<0.05), whereas not related to the age and expression of ER, PR, Ki-67, and Her-2(P>0.05); (2) the setting of FBI-1 lower expression pre-neoadjuvant chemotherapy had superior treatment outcome than the high expression setting based on either clinical assessment (86.7% vs 51.4%, P=0.027) or pathological assessment(80.0% vs 28.6%, P=0.001); (3) the rate of upregulated FBI-1 expression was significantly decreased post-neoadjuvant chemotherapy(70.0% vs 38.0%, P=0.004), with FBI-1 expression of 22 patients downregulated (62.9%); (4) the expression of FBI-1 in responded setting was significantly decreased than that in the non-responded setting based on either clinical (77.4% vs 26.3%, P=0.001) or pathological (72.7% vs 39.3%, P=0.024) assessment. The downregulation of FBI-1 was correlated to either clinical efficacy (r=0.440, P<0.01) or pathological efficacy (r=0.491, P<0.05) of neoadjuvant chemotherapy. Conclusion: In breast cancer patients receiving neoadjuvant chemotherapy, the upregulated expression of FBI-1 in breast cancer lesion is associated with clinical stage and lymph node metastasis. The neoadjuvant chemotherapy can significantly reduce the expression of FBI-1. The upregulated expression of FBI-1 may be predictive of resistance to chemotherapeutic drugs, and has predictive value for the efficacy of neoadjuvant chemotherapy in breast cancer patients.

MicroRNA-137 represses FBI-1 to inhibit proliferation and in vitro invasion and migration of hepatocellular carcinoma cells.

The pro-oncogene factor that binds to inducer of short transcripts-1 (FBI-1), which is encoded by ZBTB7A gene and belongs to POK (POZ/BTB and KrÜppel) protein family, has been shown to enhance hepatocellular carcinoma (HCC) cells proliferation and multi-drug resistance (MDR) process. However, the possibility that FBI-1 is a therapeutic target for further HCC treatment remains poorly determined. In the current study, two microRNA (miRNA) target prediction programs (TargetScan and MiRanda) were used to identify miR-137 as a potential regulator of FBI-1. Our results showed that expression of miR-137 was downregulated, while FBI-1 was upregulated in clinical HCC specimens, compared with paired non-tumor specimens. Overexpression of miR-137 via adenoviral vector inhibited the proliferation and anchorage-independent growth of HCC cells, HepG2 and MHCC-97H. Our data also showed that miR-137 repressed endogenous expression level of FBI-1, as well as Notch-1 and Survivin. MiR-137 also inhibited in vitro invasion and migration of HCC cells and attenuated their epithelial-mesenchymal transition (EMT) process. Moreover, miR-137 suppressed the growth rate of HepG2 cells in nude mice model. Overexpression of miR-137 via its adenoviral vector enhanced the sensitivity of HepG2 cells to anti-tumor drugs and attenuated the MDR process of a resistance cell line HepG2/adriamycin (ADR). Thus, FBI-1 downregulation mediated by miR-137 overexpression may be a potential strategy for HCC treatment.

Human Kruppel-related 3 (HKR3) is a novel transcription activator of alternate reading frame (ARF) gene.

HKR3 (Human Krüppel-related 3) is a novel POK (POZ-domain Krüppel-like zinc-finger) family transcription factor. Recently, some of the POK (POZ-domain Krüppel-like zinc finger) family proteins have been shown to play roles in cell cycle arrest, apoptosis, cell proliferation, and oncogenesis. We investigated whether HKR3, an inhibitor of cell proliferation and an uncharacterized POK family protein, could regulate the cell cycle by controlling expression of genes within the p53 pathway (ARF-MDM2-TP53-p21WAF/CDKN1A). HKR3 potently activated the transcription of the tumor suppressor gene ARF by acting on the proximal promoter region (bp, -149∼+53), which contains Sp1 and FBI-1 binding elements (FREs). HKR3 interacted with the co-activator p300 to activate ARF transcription, which increased the acetylation of histones H3 and H4 within the proximal promoter. Oligonucleotide pull-down assays and ChIP assays revealed that HKR3 interferes with the binding of the proto-oncogenic transcription repressor FBI-1 to proximal FREs, thus derepressing ARF transcription.

Capsaicin Inhibits Proliferation and Induces Apoptosis in Breast Cancer by Down-Regulating FBI-1-Mediated NF-κB Pathway.

BACKGROUND: As a natural compound extracted from a variety of hot peppers, capsaicin has drawn increasing attention to its anti-cancer effects against multiple human cancers including breast cancer. FBI-1 is a major proto-oncogene negatively regulating the transcription of many tumor suppressor genes, and plays a vital role in tumorigenesis and progression. However, whether FBI-1 is involved in capsaicin-induced breast cancer suppression has yet to be ascertained. This study aimed to investigate the effects of capsaicin on proliferation and apoptosis and its association with FBI-1 expression in breast cancer. METHODS: CCK-8 and morphological observation assay were employed to detect cell proliferation. Flow cytometry and TUNEL assay were conducted to detect cell apoptosis. RNA interference technique was used to overexpress or silence FBI-1 expression. qRT-PCR and/or Western blot analysis were applied to detect the protein expression of FBI-1, Ki-67, Bcl-2, Bax, cleaved-Caspase 3, Survivin and NF-κB p65. Xenograft model in nude mice was established to assess the in vivo effects. RESULTS: Capsaicin significantly inhibited proliferation and induced apoptosis in breast cancer in vitro and in vivo, along with decreased FBI-1, Ki-67, Bcl-2 and Survivin protein expression, increased Bax protein expression and activated Caspase 3. Furthermore, FBI-1 overexpression obviously attenuated the capsaicin-induced anti-proliferation and pro-apoptosis effect, accompanied with the above-mentioned proteins reversed, whereas FBI-1 silencing generated exactly the opposite response. In addition, as a target gene of FBI-1, NF-κB was inactivated by p65 nuclear translocation suppressed with capsaicin treatment, which was perceptibly weakened with FBI-1 overexpression or enhanced with FBI-1 silencing. CONCLUSION: This study reveals that FBI-1 is closely involved in capsaicin-induced anti-proliferation and pro-apoptosis of breast cancer. The underlying mechanism may be related to down-regulation of FBI-1-mediated NF-κB pathway. Targeting FBI-1 with capsaicin may be a promising therapeutic strategy in patients with breast cancer.

Hypoxia-induced RelA/p65 derepresses SLC16A3 (MCT4) by downregulating ZBTB7A.

Overexpressed Solute Carrier Family 16 Member 3 (SLC16A3, also called MCT4) plays a critical role in hypoxic cancer cell growth and proliferation, by expelling glycolysis-derived lactate across the plasma membrane. However, how SLC16A3 expression is regulated, under hypoxic conditions, is poorly understood. FBI-1, encoded by ZBTB7A, is a proto-oncoprotein. Interestingly, under hypoxic conditions, expression of SLC16A3, and hypoxia-inducible factor-1 (HIF-1), increased gradually, while FBI-1 expression decreased, suggesting a negative correlation between SLC16A3/HIF-1 and FBI-1 expression. Consequently, we hypothesized that FBI-1 might regulate SLC16A3 and/or HIF-1 expression. Transient transfection and transcription assays of SLC16A3 promoter reporter fusion constructs, oligonucleotide-pulldowns, and ChIP assays, showed that HIF-1α activates SLC16A3 by binding to a hypoxia-response element (HRE), while ectopic FBI-1 potently repressed SLC16A3, by binding to both FBI-1-response elements (FREs) and HREs, during hypoxia. Further evidence for this model was downregulation of ZBTB7A, correlated with SLC16A3 upregulation, in hypoxic colon cancer cells. We also investigated how FBI-1 expression is downregulated during hypoxia. The 5'-upstream regulatory region of ZBTB7A contains two NF-κB-binding sites and two HREs. Interestingly, hypoxia activated NF-κB (RelA/p65) and also increased its nuclear translocation. NF-κB repressed ZBTB7A by binding NF-κB-binding elements, and downregulated the repressor FBI-1, thereby increasing SLC16A3 transcription. While transcriptional repression of SLC16A3 by FBI-1 inhibited lactate efflux, repression of ZBTB7A and activation of lactate efflux by NF-κB, increased colon cancer cell growth and proliferation.

FBI-1 mRNA in normal mucosa is an independent prognostic factor in colorectal cancer patients.

Although several studies provide evidence that FBI-1 is an important gene regulator in colorectal cancer (CRC), it is noteworthy that, to our knowledge, no analysis of the correlation between FBI-1 expression and prognosis in CRC has been reported. Using real-time RT-PCR, we detected FBI-1 mRNA in 161 CRC patients (primary tumor, along with the corresponding normal mucosa), 36 liver metastases, and analyzed the relationship of its expression with clinicopathological features. Colon cancer cell lines were used to study FBI-1 function. Our study found that FBI-1 was significant up-regulated in tumor tissue (2.621 ± 0.157) compared with the corresponding normal mucosa (1.620 ± 0.165, P < 0.0001). FBI-1 in normal mucosa was a prognostic factor (P = 0.039, RR 0.431, 95% CI 0.194-0.958), independent of gender, age, stage, and differentiation. High levels of FBI-1 mRNA were related with good survival. Patients with complications had a higher primary tumor FBI-1 expression than those without complications (3.400 ± 0.332 vs. 2.516 ± 0.241, P = 0.032). Suppression of FBI-1 in colon cancer cell lines could repress proliferation of cancer cells. In conclusion, FBI-1 mRNA is overexpressed in CRC, and takes part in the development of CRC. FBI-1 mRNA in normal mucosa is an independent prognostic factor. Our findings give further support to the concept of "field cancerization", and hint that when we study a biomarker, we should not only focus on the tumor tissue but also the corresponding normal mucosa.