Lishan Fang

MicroRNA-374a activates Wnt/β-catenin signaling to promote breast cancer metastasis

Tumor metastasis involves a series of biological steps during which the tumor cells acquire the ability to invade surrounding tissues and survive outside the original tumor site. During the early stages, the cancer cells undergo an epithelial-mesenchymal transition (EMT). Wnt/β-catenin signaling is known to drive EMT and metastasis. Here we report that Wnt/β-catenin signaling is hyperactivated in metastatic breast cancer cells that express microRNA 374a (miR-374a). In breast cancer cell lines, ectopic overexpression of miR-374a promoted EMT and metastasis both in vitro and in vivo. Furthermore, miR-374a directly targeted and suppressed multiple negative regulators of the Wnt/β-catenin signaling cascade, including WIF1, PTEN, and WNT5A. Notably, miR-374a was markedly upregulated in primary tumor samples from patients with distant metastases and was associated with poor metastasis-free survival. These results demonstrate that miR-374a maintains constitutively activated Wnt/β-catenin signaling and may represent a therapeutic target for early metastatic breast cancer.

MicroRNA-30e* promotes human glioma cell invasiveness in an orthotopic xenotransplantation model by disrupting the NF-κB/IκBα negative feedback loop

Constitutive activation of NF-κB is a frequent event in human cancers, playing important roles in cancer development and progression. In nontransformed cells, NF-κB activation is tightly controlled by IκBs. IκBs bind NF-κB in the cytoplasm, preventing it from translocating to the nucleus to modulate gene expression. Stimuli that activate NF-κB signaling trigger IκB degradation, enabling nuclear translocation of NF-κB. Among the genes regulated by NF-κB are those encoding the IκBs, providing a negative feedback loop that limits NF-κB activity. How transformed cells override this NF-κB/IκB negative feedback loop remains unclear. Here, we report in human glioma cell lines that microRNA-30e* (miR-30e*) directly targets the IκBα 3ι-UTR and suppresses IκBα expression. Overexpression of miR-30e* in human glioma cell lines led to hyperactivation of NF-κB and enhanced expression of NF-κB-regulated genes, which promoted glioma cell invasiveness in in vitro assays and in an orthotopic xenotransplantation model. These effects of miR-30e* were shown to be clinically relevant, as miR-30e* was found to be upregulated in primary human glioma cells and correlated with malignant progression and poor survival. Hence, miR-30e* provides an epigenetic mechanism that disrupts the NF-κB/IκBα loop and may represent a new therapeutic target and prognostic marker.

miR-205 Targets PTEN and PHLPP2 to Augment AKT Signaling and Drive Malignant Phenotypes in Non–Small Cell Lung Cancer

AKT signaling is constitutively activated in various cancers, due in large part to loss-of-function in the PTEN and PHLPP phosphatases that act as tumor suppressor genes. However, AKT signaling is activated widely in non-small cell lung cancers (NSCLC) where genetic alterations in PTEN or PHLPP genes are rare, suggesting an undefined mechanism(s) for their suppression. In this study, we report upregulation of the oncomir microRNA (miR)-205 in multiple subtypes of NSCLC, which directly represses PTEN and PHLPP2 expression and activates both the AKT/FOXO3a and AKT/mTOR signaling pathways. miR-205 overexpression in NSCLC cells accelerated tumor cell proliferation and promoted blood vessel formation in vitro and in vivo. Conversely, RNA interference-mediated silencing of endogenous miR-205 abrogated these effects. The malignant properties induced by miR-205 in NSCLC cells were reversed by AKT inhibitors, FOXO3a overexpression, rapamycin treatment, or restoring PHLPP2 or PTEN expression. Mechanistic investigations revealed that miR-205 overexpression was a result of NF-κB-mediated transactivation of the miR-205 gene. Taken together, our results define a major epigenetic mechanism for suppression of PTEN and PHLPP2 in NSCLC, identifying a pivotal role for miR-205 in development and progression of this widespread disease.

MiR‐136 promotes apoptosis of glioma cells by targeting AEG‐1 and Bcl‐2

MicroRNAs have the capacity to coordinately repress multiple target genes and interfere with biological functions of the cell, such as proliferation and apoptosis. Here we report that miR‐136 is downregulated in human glioma, and that the miRNA promotes apoptosis of glioma cells induced by chemotherapy. Two anti‐apoptotic genes, AEG‐1 and Bcl‐2, are identified as targets of miR‐136, and restoration of AEG‐1 or Bcl‐2 expression suppresses miR‐136‐enhanced apoptosis. Therefore, miR‐136 might play a tumor‐suppressive role in human glioma and thereby might represent a potential therapeutic strategy.

miR-186 Downregulation Correlates with Poor Survival in Lung Adenocarcinoma, Where It Interferes with Cell-Cycle Regulation

Deeper mechanistic understanding of lung adenocarcinoma (non-small cell lung carcinoma, or NSCLC), a leading cause of cancer-related deaths overall, may lead to more effective therapeutic strategies. In analyzing NSCLC clinical specimens and cell lines, we discovered a uniform decrease in miR-186 (MIR186) expression in comparison with normal lung tissue or epithelial cell lines. miR-186 expression correlated with patient survival, with median overall survival time of 63.0 or 21.5 months in cases exhibiting high or low levels of miR-186, respectively. Enforced overexpression of miR-186 in NSCLC cells inhibited proliferation by inducing G(1)-S checkpoint arrest. Conversely, RNA interference-mediated silencing miR-186 expression promoted cell-cycle progression and accelerated the proliferation of NSCLC cells. Cyclin D1 (CCND1), cyclin-dependent kinase (CDK)2, and CDK6 were each directly targeted for inhibition by miR-186 and restoring their expression reversed miR-186-mediated inhibition of cell-cycle progression. The inverse relationship between expression of miR-186 and its targets was confirmed in NSCLC tumor xenografts and clinical specimens. Taken together, our findings established a tumor-suppressive role for miR-186 in the progression of NSCLC.

Aberrantly expressed miR-582-3p maintains lung cancer stem cell-like traits by activating Wnt/β-catenin signalling

Cancer stem cells (CSCs) are involved in tumorigenesis, tumour recurrence and therapy resistance and Wnt signalling is essential for the development of the biological traits of CSCs. In non-small cell lung carcinoma (NSCLC), unlike in colon cancer, mutations in β-catenin and APC genes are uncommon; thus, the mechanism underlying the constitutive activation of Wnt signalling in NSCLC remains unclear. Here we report that miR-582-3p expression correlates with the overall- and recurrence-free-survival of NSCLC patients, and miR-582-3p has an activating effect on Wnt/β-catenin signalling. miR-582-3p overexpression simultaneously targets multiple negative regulators of the Wnt/β-catenin pathway, namely, AXIN2, DKK3 and SFRP1. Consequently, miR-582-3p promotes CSC traits of NSCLC cells in vitro and tumorigenesis and tumour recurrence in vivo. Antagonizing miR-582-3p potently inhibits tumour initiation and progression in xenografted animal models. These findings suggest that miR-582-3p mediates the constitutive activation of Wnt/β-catenin signalling, likely serving as a potential therapeutic target for NSCLC.

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma

Background: MicroRNAs play important roles in regulating AKT pathway. Results: miR-542-3p suppresses glioblastoma cell invasion through directly inhibiting AKT1, ILK, and PIK3R1. Conclusion: miR-542-3p down-regulation contributes to aberrant activation of the AKT signaling, and miR-542-3p acts as a negative regulator in astrocytoma progression. Significance: Learning how miRNAs participate in AKT pathway is crucial for understanding its regulators and cancer therapy. The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma.

Simultaneous overactivation of Wnt/β-catenin and TGFβ signalling by miR-128-3p confers chemoresistance-associated metastasis in NSCLC

Cancer chemoresistance and metastasis are tightly associated features. However, whether they share common molecular mechanisms and thus can be targeted with one common strategy remain unclear in non-small cell lung cancer (NSCLC). Here, we report that high levels of microRNA-128-3p (miR-128-3p) is key to concomitant development of chemoresistance and metastasis in residual NSCLC cells having survived repeated chemotherapy and correlates with chemoresistance, aggressiveness and poor prognosis in NSCLC patients. Mechanistically, miR-128-3p induces mesenchymal and stemness-like properties through downregulating multiple inhibitors of Wnt/β-catenin and TGF-β pathways, leading to their overactivation. Importantly, antagonism of miR-128-3p potently reverses metastasis and chemoresistance of highly malignant NSCLC cells, which could be completely reversed by restoring Wnt/β-catenin and TGF-β activities. Notably, correlations among miR-128-3p levels, activated β-catenin and TGF-β signalling, and pro-epithelial-to-mesenchymal transition/pro-metastatic protein levels are validated in NSCLC patient specimens. These findings suggest that miR-128-3p might be a potential target against both metastasis and chemoresistance in NSCLC.

URGCP promotes non-small cell lung cancer invasiveness by activating the NF-κB-MMP-9 pathway

Invasion and metastasis are main traits of tumor progression and responsible for the poor prognosis of advanced non-small cell lung cancer (NSCLC). The molecular mechanisms underlying the malignant behaviors of NSCLC remain incompletely understood. The present study demonstrate that up-regulator of cell proliferation (URGCP), a recently identified tumor-promoting gene found in several tumor types, is markedly overexpressed in human NSCLC cell lines and clinical NSCLC samples. URGCP upregulation correlates significantly with the progression and poor prognosis of this disease. In vitro and in vivo studies demonstrate that increasing URGCP expression accelerates invasion, migration, and distant metastasis of NSCLC cells whereas downregulating URGCP suppresses these malignant traits. Notably, silencing URGCP expression almost completely abrogates the metastatic ability of NSCLC cells. At the molecular level, URGCP markedly promotes MMP-9 expression by activating NF-κB signaling. Additionally, URGCP and MMP-9 expression are positively correlated in various cohorts of human NSCLC specimens, and NF-κB-activated MMP-9 expression contributes to URGCP-induced invasiveness of NSCLC cell lines. Collectively, these findings indicate that URGCP plays an important role in promoting NSCLC cell invasion and metastasis by enhancing NF-κB-activated MMP-9 expression and may serve as a potential therapeutic target and prognostic marker.

CK1α suppresses lung tumour growth by stabilizing PTEN and inducing autophagy

The contribution of autophagy to cancer development remains controversial, largely owing to the fact that autophagy can be tumour suppressive or oncogenic in different biological contexts. Here, we show that in non-small-cell lung cancer (NSCLC), casein kinase 1 alpha 1 (CK1α) suppresses tumour growth by functioning as an autophagy inducer to activate an autophagy-regulating, tumour-suppressive PTEN/AKT/FOXO3a/Atg7 axis. Specifically, CK1α bound the C-terminal tail of PTEN and enhanced both PTEN stability and activity by competitively antagonizing NEDD4-1-induced PTEN polyubiquitination and abrogating PTEN phosphorylation, thereby inhibiting AKT activity and activating FOXO3a-induced transcription of Atg7. Notably, blocking CK1α-induced Atg7-dependent autophagy cooperates with oncogenic HRasV12 to initiate tumorigenesis of lung epithelial cells. An association of a CK1α-modulated autophagic program with the anti-neoplastic activities of the CK1α/PTEN/FOXO3a/Atg7 axis was demonstrated in xenografted tumour models and human NSCLC specimens. This provides insights into the biological and potentially clinical significance of autophagy in NSCLC.Cai et al. show that CK1α suppresses lung cancer growth by inducing autophagy through a PTEN–AKT–FOXO3a pathway that ultimately leads to ATG7 expression.