NUCB2 promotes hepatocellular carcinoma cell growth and metastasis by activating the E2F4/PTGR1 axis.

Background: The important role of nucleobindin 2 (NUCB2) in various cancers has been recently recognized. However, its biological functions and regulatory mechanisms in hepatocellular carcinoma (HCC) remain unclear. Methods: The expression level of NUCB2 in HCC was assessed using public databases, immunohistochemistry, and Western blotting. The effects of NUCB2 on cell proliferation and metastasis were investigated using colony formation, EdU, Transwell assays, and an in vivo mouse xenograft model. Regulation of E2F4 by NUCB2 was identified by protein half-life and in vivo ubiquitylation assays. The relationship between E2F4 and prostaglandin reductase 1 (PTGR1) was investigated by qRT-PCR, RT-PCR, and chromatin immunoprecipitation assays. Results: This study found that NUCB2 expression was significantly higher in HCC tissues than in normal liver tissues, and patients with high expression displayed shorter survival rates. Inhibition of NUCB2 reduced the proliferation and metastatic potential of HCC cells in vitro and in vivo. NUCB2 depletion reduced PTGR1 expression, which reduced cell proliferation and migration. Our findings suggested that NUCB2 suppressed E2F4 degradation by interacting with E2F4. Additionally, increased E2F4 levels facilitated PTGR1 transcription by directly binding to the PTGR1 promoter. Conclusion: This study demonstrated the oncogenic properties of NUCB2 in HCC and suggested that NUCB2 facilitates hepatocellular carcinoma progression by activating the E2F4/PTGR1 axis.

KLF4 suppresses anticancer effects of brusatol via transcriptional upregulating NCK2 expression in melanoma.

Brusatol (Bru), a main extract from traditional Chinese medicine Brucea javanica, has been reported to exist antitumor effect in many tumors including melanoma. However, the underlying mechanism in its anti-melanoma effect still need further exploration. Here, we reported that the protein expression of KLF4 in melanoma cells were significantly downregulated in response to brusatol treatment. Overexpression of KLF4 suppressed brusatol-induced melanoma cell apoptosis; while knockdown of KLF4 enhanced antitumor effects of brusatol on melanoma cells not only in vitro but also in vivo. Further studies on the mechanism revealed that KLF4 bound to the promoter of NCK2 directly and facilitated NCK2 transcription, which suppressed the antitumor effect of brusatol on melanoma. Furthermore, our findings showed that miR-150-3p was dramatically upregulated under brusatol treatment which resulted in the downregulation of KLF4. Our results suggested that the miR-150-3p/KLF4/NCK2 axis might play an important role in the antitumour effects of brusatol in melanoma.

HOXB9 promotes osteosarcoma cell survival and malignancy under glucose starvation via upregulating SPP1 expression.

Homeobox B9 (HOXB9) has been shown to play a critical role in several tumors. However, the precise biological mechanisms and functions of HOXB9 in osteosarcoma remain largely unknown. In this study, we found that HOXB9 was increased upon glucose starvation. Elevated HOXB9 suppressed osteosarcoma cell death and supported cell growth and migration under glucose starvation. Further mechanistic studies demonstrated that HOXB9 directly bound to the promoter of secreted phosphoprotein 1 (SPP1) and transcriptionally upregulated SPP1 expression which then led cell death decrease and cell growth increase under glucose deprivation environment. Clinically, HOXB9 was significantly upregulated in osteosarcoma compared with normal tissues and increase of HOXB9 expression was positively associated with the elevation of SPP1 in osteosarcoma. Overall, our study illustrates that HOXB9 contributes to malignancy in osteosarcoma and inhibits cell death through transcriptional upregulating SPP1 under glucose starvation.

NUCB2 inhibition antagonizes osteosarcoma progression and promotes anti-tumor immunity through inactivating NUCKS1/CXCL8 axis.

The oncogenic properties of Nucleobindin2 (NUCB2) have been observed in various cancer types. Nevertheless, the precise understanding of the biological functions and regulatory mechanisms of NUCB2 in osteosarcoma remains limited. This investigation reported that NUCB2 was significantly increased upon glucose deprivation-induced metabolic stress. Elevated NUCB2 suppressed glucose deprivation-induced cell death and reactive oxygen species (ROS) increase. Depletion of NUCB2 resulted in a reduction in osteosarcoma cell proliferation as well as metastatic potential in vitro and in vivo. Mechanically, NUCB2 ablation suppressed C-X-C Motif Chemokine Ligand 8 (CXCL8) expression which then reduced programmed cell death 1 ligand 1 (PD-L1) expression and stimulated anti-tumor immunity mediated through cytotoxic T cells. Importantly, a combination of NUCB2 depletion with anti-PD-L1 treatment improved anti-tumor T-cell immunity in vivo. Moreover, we further demonstrated that NUCB2 interacted with NUCKS1 to inhibit its degradation, which is responsible for the transcriptional regulation of CXCL8 expression. Altogether, the outcome emphasizes the function of NUCB2 in osteosarcoma and indicates that NUCB2 elevates osteosarcoma progression and immunosuppressive microenvironment through the NUCKS1/CXCL8 pathway.

LINC00629, a HOXB4-downregulated long noncoding RNA, inhibits glycolysis and ovarian cancer progression by destabilizing c-Myc.

Ovarian cancer (OC) cells typically reprogram their metabolism to promote rapid proliferation. However, the role of long noncoding RNAs (lncRNAs) in the metabolic reprogramming of ovarian cancer, especially in glucose metabolic reprogramming, remains largely unknown. LINC00629 has been reported in our previous study to promote osteosarcoma progression. Upregulated LINC00629 was found to enhance the growth-suppressive effect of apigenin on oral squamous cell carcinoma. However, the precise function of LINC00629 in ovarian cancer development remains poorly understood. In this study, we found that LINC00629 was significantly downregulated in OC tissues and that low LINC00629 expression was associated with poor survival. Inhibition of LINC00629 was required for increased glycolysis activity and cell proliferation in ovarian cancer. In vivo, overexpression of LINC00629 dramatically inhibited tumor growth and lung metastasis. Mechanistically, LINC00629 interacted with and destabilized c-Myc, leading to its ubiquitination and proteasome degradation, further resulting in increased expression of downstream glycolysis-related genes and glucose metabolic reprogramming in OC. Interestingly, HOXB4 bound to the LINC00629 promoter and inhibited its transcription, indicating that LINC00629 is a transcriptional target of HOXB4. Collectively, these findings establish a direct role for LINC00629 in suppressing glucose metabolism, and HOXB4/LINC00629/c-Myc might serve as a potential biomarker and an effective therapeutic strategy for OC cancer treatment.

NUCKS1, a LINC00629-upregulated gene, facilitated osteosarcoma progression and metastasis by elevating asparagine synthesis.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) has been reported to play an oncogenic role in several cancers. However, the biological functions and regulatory mechanism of NUCKS1 in osteosarcoma have not been fully understood. In this study, we reported that NUCKS1 was significantly increased in osteosarcoma. Depletion of NUCKS1 decreased osteosarcoma cell proliferation and metastasis in vivo and in vitro. Overexpression of NUCKS1 accelerated osteosarcoma cell aggressiveness. Mechanistically, NUCKS1 facilitated asparagine (Asn) synthesis by transcriptionally upregulating asparagine synthetase (ASNS) expression and elevating the levels of Asn in osteosarcoma cells, leading to increased cell growth and metastasis. Inhibition of ASNS or reduction of Asn decreased osteosarcoma cell aggressiveness and impaired the promoting effects of NUCKS1 on tumorigenesis and metastasis. Furthermore, we also found that by acting as a sponge for miR-4768-3p, LINC00629 promoted NUCKS1 expression. Collectively, our findings highlight the role of NUCKS1 in regulating asparagine metabolism and reveal that LINC00629 is an important regulator of NUCKS1 that contributes to NUCKS1 upregulation in osteosarcoma.

SNHG1, a KLF4-upregulated gene, promotes glioma cell survival and tumorigenesis under endoplasmic reticulum stress by upregulating BIRC3 expression.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the resistance to endoplasmic reticulum (ER) stress in many cancers. However, ER stress-regulated lncRNAs are still unknown in glioma. In the present study, we investigated the altered lncRNAs upon ER stress in glioma and found that small nucleolar RNA host gene 1 (SNHG1) was markedly increased in response to ER stress. Increased SNHG1 suppressed ER stress-induced apoptosis and promoted tumorigenesis in vitro and in vivo. Further mechanistic studies indicated that SNHG1 elevated BIRC3 mRNA stability and enhanced BIRC3 expression. We also found that KLF4 transcriptionally upregulated SNHG1 expression and contributed to the ER stress-induced SNHG1 increase. Collectively, the present findings indicated that SNHG1 is a KLF4-regulated lncRNA that suppresses ER stress-induced apoptosis and facilitates gliomagenesis by elevating BIRC3 expression.

Erratum: KLF4 suppresses the migration of hepatocellular carcinoma by transcriptionally upregulating monoglyceride lipase.

[This corrects the article on p. 1019 in vol. 8, PMID: 30034939.].

HOXB9 a miR-122-5p regulated gene, suppressed the anticancer effects of brusatol by upregulating SCD1 expression in melanoma.

Brusatol (Bru), a Chinese medicine Brucea javanica extract, has a variety of antitumour effects. However, its role and underlying mechanism in melanoma have not been fully elucidated. In this study, we found that brusatol inhibited melanoma cell proliferation and migration and promoted cell apoptosis in vitro, in addition to suppressing melanoma cell tumorigenesis in vivo. Further studies on the mechanism revealed that brusatol significantly downregulated the expression of stearoyl-CoA desaturase 1 (SCD1). Increased SCD1 expression could impair the antitumour effects of brusatol on melanoma cells. Subsequently, we found that HOXB9, an important transcription factor, was directly bound to the promoter of SCD1, facilitating its transcription. Overexpression of HOXB9 inhibited brusatol-induced SCD1 reduction and promoted cell survival. Furthermore, our results revealed that miR-122-5p was significantly increased in response to brusatol treatment and led to a decrease in HOXB9 in melanoma. Collectively, our data suggested that the miR-122-5p/HOXB9/SCD1 axis might play an important role in the antitumour effects of brusatol and that brusatol might have potential clinical implications in melanoma therapy.

LINC00629 protects osteosarcoma cell from ER stress-induced apoptosis and facilitates tumour progression by elevating KLF4 stability.

BACKGROUND: Escaping from ER stress-induced apoptosis plays an important role in the progression of many tumours. However, its molecular mechanism in osteosarcoma remains incompletely understood. METHODS: The molecular mechanism was investigated using RNA sequencing, qRT-PCR and Western blot assays. The relationship between LINC00629 and KLF4 was investigated using RNA pulldown and ubiquitylation assays. The transcriptional regulation of laminin subunit alpha 4 (LAMA4) by KLF4 was identified using bioinformatic analysis, a luciferase assay, and a chromatin immunoprecipitation assay. RESULTS: Here, we demonstrated that LINC00629 was increased under ER stress treatment. Elevated LINC00629 inhibited ER stress-induced osteosarcoma cell apoptosis and promoted clonogenicity and migration in vitro and in vivo. Further mechanistic studies indicated that LINC00629 interacted with KLF4 and suppressed its degradation, which led to a KLF4 increase in osteosarcoma. In addition, we also found that KLF4 upregulated LAMA4 expression by directly binding to its promoter and that LINC00629 inhibited ER stress-induced apoptosis and facilitated osteosarcoma cell clonogenicity and metastasis by activating the KLF4-LAMA4 pathway. CONCLUSION: Collectively, our data indicate that LINC00629 is a critical long noncoding RNA (lncRNA) induced by ER stress and plays an oncogenic role in osteosarcoma cell by activating the KLF4-LAMA4 axis.

LINC00629, a KLF10-responsive lncRNA, promotes the anticancer effects of apigenin by decreasing Mcl1 stability in oral squamous cell carcinoma.

Apigenin, a naturally occurring flavonoid, is known to exhibit antitumor activity in many cancers. However, the regulatory mechanism of apigenin and the long noncoding RNAs (lncRNAs) altered upon apigenin treatment in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we found that LINC00629 was significantly upregulated in response to apigenin treatment. Upregulated LINC00629 enhanced the growth-suppressive and proapoptotic effects of apigenin on OSCC cells by interacting with Mcl1 and facilitating its degradation. Subsequently, our data indicated that KLF10, an important transcription factor, directly bound to the promoter of LINC00629, facilitating its transcription and contributing to apigenin-induced LINC00629 expression. Collectively, these results suggest that the KLF10-LINC00629-Mcl1 axis plays an important role in the anticancer effects of apigenin.

ZBTB7A, a miR-144-3p targeted gene, accelerates bladder cancer progression via downregulating HIC1 expression.

BACKGROUND: Zinc finger and BTB domain-containing 7A (ZBTB7A) is a member of the POK family of transcription factors that plays an oncogenic or tumor-suppressive role in different cancers depending on the type and genetic context of cancer. However, the function and molecular mechanism of ZBTB7A in bladder cancer (BC) remain elusive. METHODS: The role of ZBTB7A in bladder cancer was detected by colony formation, transwell, and tumor formation assays. The expression levels of ZBTB7A, HIC1, and miR-144-3p were analyzed by qRT-PCR and Western blot. Bioinformatics analysis and a dual-luciferase reporter assay were used to assess the effect of ZBTB7A on the promoter activity of HIC1. RESULTS: The present study revealed that knockdown of ZBTB7A suppressed BC cell growth and migration, as indicated by an approximately 50% reduction in the number of colonies and an approximately 70% reduction in the number of migrated cells. Loss of ZBTB7A inhibited tumor growth in vivo, resulting in a 75% decrease in tumor volume and an 80% decrease in tumor weight. Further mechanistic studies revealed that ZBTB7A bound to the hypermethylated in cancer 1 (HIC1) promoter and downregulated HIC1 expression, accelerating the malignant behavior of BC. Increased expression of ZBTB7A in BC tissues was negatively corrected with the expression of HIC1. Moreover, ZBTB7A was a target of miR-144-3p, which decreased ZBTB7A expression in BC. CONCLUSION: Our data demonstrate that ZBTB7A, a targeted gene of miR-144-3p, promoted tumorigenesis of BC through downregulating HIC1 expression.

ITIH5, a p53-responsive gene, inhibits the growth and metastasis of melanoma cells by downregulating the transcriptional activity of KLF4.

ITIH5, a member of the inter-α-trypsin inhibitory (ITI) gene family, acts as a putative tumour-suppressor gene in many cancers. However, its role and the regulatory mechanism in melanoma are still unclear. Here, we found that the expression of ITIH5 was decreased in melanoma tissues compared with normal skin tissues. Decreased expression of ITIH5 was correlated with clinicopathological features and predicted poor prognosis in patients with melanoma. Forced expression of ITIH5 significantly inhibited melanoma cell proliferation and metastasis in vitro and ex vivo while knockdown of ITIH5 expression enhanced the malignant behaviour of melanoma cells. In further mechanistic studies, we showed that p53 can directly bind to the promoter of ITIH5 and thus promotes transcription of ITIH5 in melanoma cells. Additionally, we found that ITIH5 interacted with Krüppel-like factor 4 (KLF4) and inhibited its transcriptional activity. Collectively, our data not only identified a tumour-suppressive role of ITIH5 in melanoma but also revealed that upregulation of ITIH5 by p53 suppressed melanoma cell growth and migration likely by downmodulating the transcriptional activity of KLF4.

Sialyltransferase7A promotes angiotensin II-induced cardiomyocyte hypertrophy via HIF-1α-TAK1 signalling pathway.

AIMS: Sialylation is up-regulated during the development of cardiac hypertrophy. Sialyltransferase7A (Siat7A) mRNA is consistently over-expressed in the hypertrophic left ventricle of hypertensive rats independently of genetic background. The aims of this study were: (i) to detect the Siat7A protein levels and its roles in the pathological cardiomyocyte hypertrophy; (ii) to elucidate the effect of sialylation mediated by Siat7A on the transforming-growth-factor-ß-activated kinase (TAK1) expression and activity in cardiomyocyte hypertrophy; and (iii) to clarify hypoxia-inducible factor 1 (HIF-1) expression was regulated by Siat7A and transactivated TAK1 expression in cardiomyocyte hypertrophy. METHODS AND RESULTS: Siat7A protein level was increased in hypertrophic cardiomyocytes of human and rats subjected to chronic infusion of angiotensin II (ANG II). Delivery of adeno-associated viral (AAV9) bearing shRNA against rat Siat7A into the left ventricular wall inhibited ventricular hypertrophy. Cardiac-specific Siat7A overexpression via intravenous injection of an AAV9 vector encoding Siat7A under the cardiac troponin T (cTNT) promoter aggravated cardiac hypertrophy in ANG II-treated rats. In vitro, Siat7A knockdown inhibited the induction of Sialyl-Tn (sTn) antigen and cardiomyocyte hypertrophy stimulated by ANG II. Mechanistically, ANG II induced the activation of TAK1-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in parallel to up-regulation of Siat7A in hypertrophic cardiomyocytes. Siat7A knockdown inhibited activation of TAK1-NF-κB pathway. Interestingly, HIF-1α expression was increased in cardiomyocytes stimulated by ANG II but decreased after Siat7A knockdown. HIF-1α knockdown efficiently decreased TAK1 expression. ChIP and luciferase assays showed that HIF-1α transactivated the TAK1 promoter region (nt -1285 to -1274 bp) in the cardiomyocytes following ANG II stimulus. CONCLUSION: Siat7A was up-regulated in hypertrophic myocardium and promoted cardiomyocyte hypertrophy via activation of the HIF-1α-TAK1-NF-κB pathway.

The deubiquitinase USP10 regulates KLF4 stability and suppresses lung tumorigenesis.

Krüppel-like factor 4 (KLF4), a key transcription factor, acts as a multifunctional player involved in the progression of numerous aggressive cancers. The proteasome-dependent pathway is one of the main modalities in controlling KLF4 abundance at a posttranslational level. Although some of the ubiquitin ligases have been identified, the deubiquitinases of KLF4 and the regulatory function remain unexplored. Here, by screening ubiquitin-specific proteases that may interact with KLF4, we found ubiquitin-specific peptidase 10 (USP10) as a deubiquitinating enzyme for KLF4. Forced expression of USP10 remarkably increases KLF4 protein level by blocking the latter degradation, whereas the depletion of USP10 promotes KLF4 degradation and thus enhances tumorigenesis. Loss of USP10 in mice downregulates KLF4 expression and accelerates KrasG12D-driven lung adenocarcinoma initiation and progression. In addition, our data revealed that KLF4 can facilitate the transcription of tumor suppressor TIMP3 by directly binding to the TIMP3 promoter. Clinically, reduction of USP10 expression, concomitant with decreased KLF4 and TIMP3 abundance in carcinoma tissue, predicts poor prognosis of lung cancer patient. Taken together, our results demonstrate that USP10 is a critical regulator of KLF4, pinpointing USP10-KLF4-TIMP3 axis as a promising therapeutic target in lung cancer.