Decreased TUSC3 Promotes Pancreatic Cancer Proliferation,Invasion and Metastasis

Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.     

TUSC1, a Putative Tumor Suppressor Gene,Reduces Tumor Cell Growth In Vitro and Tumor Growth In Vivo

We previously reported the identification of TUSC1 (Tumor Suppressor Candidate 1), as a novel intronless gene isolated from a region of homozygous deletion at D9S126 on chromosome 9p in human lung cancer. In this study, we examine the differential expression of TUSC1 in human lung cancer cell lines by western blot and in a primary human lung cancer tissue microarray by immunohistochemical analysis. We also tested the functional activities and mechanisms of TUSC1 as a tumor suppressor gene through growth suppression in vitro and in vivo. The results showed no expression of TUSC1 in TUSC1 homozygously deleted cells and diminished expression in some tumor cell lines without TUSC1 deletion. Interestingly, the results from a primary human lung cancer tissue microarray suggested that higher expression of TUSC1 was correlated with increased survival times for lung cancer patients. Our data demonstrated that growth curves of tumor cell lines transfected with TUSC1 grew slower in vitro than those transfected with the empty vector. More importantly, xenograph tumors in nude mice grew significantly slower in vivo in cells stably transfected with TUSC1 than those transfected with empty vector. In addition, results from confocal microscopy and immunohistochemical analyses show distribution of TUSC1 in the cytoplasm and nucleus in tumor cell lines and in normal and tumor cells in the lung cancer tissue microarray. Taken together, our results support TUSC1 has tumor suppressor activity as a candidate tumor suppressor gene located on chromosome 9p.     

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.     

Tumor suppressor candidate 5 (TUSC5) is expressed in brown adipocytes

Rat brain endothelial cell derived gene-1 (BEC-1) had considerable homology with tumor suppressor candidate 5 (TUSC5). TUSC5 was expressed abundantly, and its mRNA was inhibited by cold exposure in rat brown adipose tissue (BAT). In the present study, we investigated its regulatory mechanism using primary cultured rat brown preadipocytes (RBPA) and Zucker lean rats (ZL). We found that: (1) TUSC5 mRNA began to increase in a manner similar to C/EBP-alpha, PPAR-gamma, and adiponectin during differentiation in RBPA; (2) neither beta3-adrenoceptor agonist BRL 37344 nor dexamethasone affected TUSC5 mRNA in RBPA; (3) propranolol did not block the decrease of TUSC5 mRNA by cold exposure in ZL; (4) BRL 37344 did not influence TUSC5 mRNA in ZL; and (5) dexamethasone inhibited TUSC5 mRNA in a dose-dependent manner similar to UCP-1 in ZL. These data suggested that TUSC5 is involved in the differentiation, and its expression is regulated independently of the beta-adrenergic pathway in BAT.     

Epidermal Growth Factor (EGF)-enhanced Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression Promotes Macrophage and Glioblastoma Cell Interaction and Tumor Cell Invasion

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCϵ- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.     

Functional Profiling of Precursor MicroRNAs Identifies MicroRNAs Essential for Glioma Proliferation

Cancer initiation and progression involve microRNAs that can function like tumor suppressors and oncogenes. The functional significance of most miRNAs is currently unknown. To determine systematically which microRNAs are essential for glioma growth, we screened a precursor microRNA library in three human glioblastoma and one astroglial cell line model systems. The most prominent and consistent cell proliferation–reducing hits were validated in secondary screening with an additional apoptosis endpoint. The functional screening data were integrated in the miRNA expression data to find underexpressed true functional tumor suppressor miRNAs. In addition, we used miRNA-target gene predictions and combined siRNA functional screening data to find the most probable miRNA-target gene pairs with a similar functional effect on proliferation. Nine novel functional miRNAs (hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a/b) in GBM cell lines were validated for their importance in glioma cell growth, and similar effects for six target genes (ROCK1, RHOA, MET, CSF1R, EIF2AK1, FGF7) of these miRNAs were shown functionally. The clinical significance of the functional hits was validated in miRNA expression data from the TCGA glioblastoma multiforme (GBM) tumor cohort. Five tumor suppressor miRNAs (hsa-miR-136, -145, -342, -129, -376a) showed significant underexpression in clinical GBM tumor samples from the TCGA GBM cohort further supporting the role of these miRNAs in vivo. Most importantly, higher hsa-miR-145 expression in GBM tumors yielded significantly better survival (p<0.005) in a subset of patients thus validating it as a genuine tumor suppressor miRNA. This systematic functional profiling provides important new knowledge about functionally relevant miRNAs in GBM biology and may offer new targets for treating glioma.     

TUSC3: a novel tumour suppressor gene and its functional implications

The tumour suppressor candidate 3 (TUSC3) gene is located on chromosome region 8p22 and encodes the 34 kD TUSC3 protein, which is a subunit of the oligosaccharyl transferase responsible for the N‐glycosylation of nascent proteins. Known to be related to autosomal recessive mental retardation for several years, TUSC3 has only recently been identified as a potential tumour suppressor gene. Based on the structure and function of TUSC3, specific mechanisms in various diseases have been investigated. Several studies have demonstrated that TUSC3 is an Mg²⁺‐transporter involved in magnesium transport and homeostasis, which is important for learning and memory, embryonic development and testis maturation. Moreover, dysfunction or deletion of TUSC3 exerts its oncological effects as a modulator by inhibiting glycosylation efficiency and consequently inducing endoplasmic reticulum stress and malignant cell transformation. In this study, we summarize the advances in the studies of TUSC3 and comment on the potential roles of TUSC3 in diagnosis and treatment of TUSC3‐related diseases, especially cancer.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

Heterogeneous Nuclear Ribonucleoprotein C1/C2 Controls the Metastatic Potential of Glioblastoma by Regulating PDCD4

MicroRNAs (miRNAs) have been implicated in the pathogenesis and progression of brain tumors. miR-21 is one of the most highly overexpressed miRNAs in glioblastoma multiforme (GBM), and its level of expression correlates with the tumor grade. Programmed cell death 4 (PDCD4) is a well-known miR-21 target and is frequently downregulated in glioblastomas in accordance with increased miR-21 expression. Downregulation of miR-21 or overexpression of PDCD4 can inhibit metastasis. Here, we investigate the role of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) in the metastatic potential of the glioblastoma cell line T98G. hnRNPC bound directly to primary miR-21 (pri-miR-21) and promoted miR-21 expression in T98G cells. Silencing of hnRNPC lowered miR-21 levels, in turn increasing the expression of PDCD4, suppressing Akt and p70S6K activation, and inhibiting migratory and invasive activities. Silencing of hnRNPC reduced cell proliferation and enhanced etoposide-induced apoptosis. In support of a role for hnRNPC in the invasiveness of GBM, highly aggressive U87MG cells showed higher hnRNPC expression levels and hnRNPC abundance in tissue arrays and also showed elevated levels as a function of brain tumor grade. Taken together, our data indicate that hnRNPC controls the aggressiveness of GBM cells through the regulation of PDCD4, underscoring the potential usefulness of hnRNPC as a prognostic and therapeutic marker of GBM.     

Suppression of tumorigenicity by MicroRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.     

LncRNA TUSC8 inhibits the invasion and migration of cervical cancer cells via miR‐641/PTEN axis

Cervical cancer (CC) is the fourth leading cause of cancer‐related death in women worldwide. There is an urgent need to find novel targets for the treatment of CC. Recently, microRNA have emerged as critical factors in tumorigenesis. In this study, we aimed to investigate the mechanism of miR‐641 on the migration and invasion of CC cells. In silico analysis revealed putative interaction between miR‐641 and phosphatase and tensin homolog (PTEN) RNA/lncRNA tumor suppressor candidate 8 (TUSC8). Hence we evaluated the expression of TUSC8, miR‐641, and PTEN. We found that the expressions of TUSC8 and PTEN were decreased in CC tissues, whereas miR‐641 expression was increased. Inhibition of miR‐641 suppressed the migration and invasion of Hela cells. In addition, TUSC8 and PTEN were upstream and downstream target molecule of miR‐641, respectively. Overexpression of TUSC8 promoted PTEN expression, and suppressed the invasion and migration of Hela cells, whereas miR‐641 mimic treatment changed the effects. These results demonstrated that overexpression of TUSC8 could inhibit the invasion and migration of CC cells by upregulating PTEN via miR‐641.     

NDUFA4L2 promotes glioblastoma progression,is associated with poor survival,and can be effectively targeted by apatinib

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.Subject terms: CNS cancer, Mitophagy