Upregulated KDM4B promotes prostate cancer cell proliferation by activating autophagy

Castration-resistant prostate cancer (CRPC) causes most of the deaths in patients with prostate cancer (PCa). The androgen receptor (AR) axis plays an important role in castration resistance. Emerging studies showed that the lysine demethylase KDM4B is a key molecule in AR signaling and turnover, and autophagy plays an important role in CRPC. However, little is known about whether KDM4B promotes CRPC progression by regulating autophagy. Here we used an androgen-independent LNCaP (LNCaP-AI) cell line to assay aberrant KDM4B expression using qPCR and western blot analysis and investigated the function of KDM4B in regulating cell proliferation. We found that KDM4B was markedly increased in LNCaP-AI cells compared with LNCaP cells. KDM4B level was significantly correlated with the Gleason score in PCa tissues. In vitro, KDM4B overexpression in CRPC cells promoted cell proliferation, whereas knockdown of KDM4B significantly inhibited cell proliferation. Upregulated KDM4B contributed to activate Wnt/β-catenin signaling and autophagy. Moreover, KDM4B activated autophagy by regulating the Wnt/β-catenin signaling. Finally, we demonstrated that autophagy inhibition attenuated KDM4B-induced CRPC cell proliferation. Our results provided novel insights into the function of KDM4B-driven CRPC development and indicated that KDM4B may be served as a potential target for CRPC therapy.     

Apolipoprotein C1 promotes prostate cancer cell proliferation in vitro

Here, we aimed to investigate the carcinogenic effects of apolipoprotein C1 (APOC1) in prostate cancer (PCa). APOC1 expression was evaluated in PCa and normal prostate specimens, and lentivirus‐mediated RNA interference was used to knockdown APOC1 in DU145 cells. The effects of APOC1 silencing on cell proliferation, cell cycle arrest, and apoptosis were assessed. APOC1 expression was much higher in PCa tissues than in normal tissues. Moreover, APOC1 silencing inhibited cell proliferation and colony formation, arrested cell cycle progression, and enhanced apoptosis in DU145 cells. Additionally, APOC1 silencing decreased survivin, phospho‐Rb, and p21 levels and increased cleaved caspase‐3 expression. These data supported the procarcinogenic effects of APOC1 in the pathogenesis of PCa and suggested that targeting APOC1 may have applications in the treatment of PCa.     

Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.     

miR-301a promotes pancreatic cancer cell proliferation by directly inhibiting bim expression

It is well known that microRNAs (miRNAs) play an important role in many diseases, including tumorigenesis. However, the mechanisms by which miRNAs regulate pancreatic cancer (PC) development remain poorly understood. In the present study, we assayed expression level of miR‐301a in PC tissues by real‐time PCR, and defined the target gene and biological function by luciferase reporter assay and Western blot analysis. We first verified that the expression level of miR‐301a was significantly increased in PC tissues. Moreover, miR‐301a overexpression promoted PC cell proliferation, whereas its depletion decreased cell proliferation. We further demonstrated that miR‐301a directly targeted 3′‐UTR of Bim gene, and inhibited its protein expression in vitro and in vivo. Importantly, Bim re‐expression reduced PC cell proliferation induced by miR‐301a. These data suggest an important role of miR‐301a in the molecular etiology of PC and implicate the potential application of miR‐301a in PC therapy. J. Cell. Biochem. 113: 3229–3235, 2012. © 2012 Wiley Periodicals, Inc.     

Induction of type XVI collagen expression facilitates proliferation of oral cancer cells

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6 h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype.     

METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer

N6-methyladenosine (m6A) is a well-known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT-qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down-regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock-down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16-mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.     

Pancreatic cancer cells respond to type I collagen by inducing snail expression to promote membrane type 1 matrix metalloproteinase-dependent collagen invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

Down-regulation of the ErbB3 binding protein 1 in human bladder cancer promotes tumor progression and cell proliferation

The ErbB3 binding protein 1 (Ebp1) represents a downstream effector of the ErbB signaling network and has been demonstrated to be a potent tumor suppressor in various human malignancies, however, its involvement in human bladder cancer is still unclear.To investigate the clinical significance and potential role of ErbB3 binding protein 1 (Ebp1) in bladder cancer. Ebp1 expression at protein and gene levels in 52 surgically removed bladder cancer specimens as well as 21 adjacent normal bladder specimens were respectively detected by immunohistochemistry and qRT-PCR. The association of Ebp1 protein expression with the clinicopathological features of bladder cancer was also statistically analyzed. Its roles in bladder cancer cell line were further evaluated. The expression level of Ebp1 protein and gene in bladder cancer tissues was significantly lower than that in adjacent normal bladder tissues (P < 0.01). When categorized into low vs. high expression, the down-regulation of Ebp1 protein was associated with the advanced pathologic stage (P = 0.036) and the high histologic grade (P = 0.001) of patients with bladder cancer. Moreover, following the transfection of Ebp1 in bladder cancer cells, not only cell proliferation and cell invasion decreased significantly, but also the cell cycle was blocked at G0/G1 stage. Our data suggest for the first time that the down-regulation of Ebp1 closely correlates with advanced clinicopathological characteristics of human bladder cancer. Furthermore, Ebp1 plays an important role in the bladder cancer cells’ proliferation by regulating the cancer cell cycle from G0/G1 to S.     

Exosome circ_0044516 promotes prostate cancer cell proliferation and metastasis as a potential biomarker

Circular RNAs (circRNAs) are important regulators in cancer growth and progression. Exosomes carry various molecules including RNA, protein, and lipid from one cell to another cell. But the role of circRNAs from the exosomes from prostate cancer patients are not elucidated. In this study, circ_0044516 was found upregulated in prostate cancer and the roles and molecular mechanism of Hsa_circ_0044516 (circ_0044516) was investigated. Firstly, the exosomes of prostate cancer patients were collected for human circRNAs microarray to screen the circRNA expression profile. There were 35 significantly expressed circRNAs with more than fivefolds from microarray analysis. Circ_0044516 was verified to be significantly upregulated in the exosomes from prostate cancer patients and the cell lines. Further investigation demonstrated that circ_0044516 downregulation inhibited the proliferation and metastasis of prostate cancer cells. By bioinformatics and luciferase reporter assays, circ_0044516 was verified to downregulate miR-29a-3p expression and negatively related to miR-29a-3p expression levels in prostate cancer. In a summary, the study indicated that circ_0044516 played an important role in prostate cancer cell survival and metastasis, which suggested that an oncogenic role of circ_0044516 in prostate cancer.     

MicroRNA-141 promotes the proliferation of non-small cell lung cancer cells by regulating expression of PHLPP1 and PHLPP2

The dysregulation of microRNAs (miRNAs) is crucially implicated in the development of various cancers. In this study, we explored the biological role of miR-141 in non-small cell lung cancer (NSCLC). miR-141 expression was significantly up-regulated in NSCLC tissues, and its overexpression accelerated NSCLC cell proliferation in vitro and tumor growth in vivo. We subsequently identified the antagonists of PI3K/AKT signaling, PH domain leucine-rich-repeats protein phosphatase 1 (PHLPP1) and PHLPP2, as direct targets of miR-141. Re-introduction of PHLPP1 and PHLPP2 abrogated miR-141-induced proliferation of NSCLC cells. Together, the results of this study suggest that miR-141 and its targets PHLPP1 and PHLPP2 play critical roles in NSCLC tumorigenesis, and provide potential therapeutic targets for NSCLC treatment.     

Down-regulation of GPR137 expression inhibits proliferation of colon cancer cells

G protein-coupled receptors （GPRs） are highly related to oncogenesis and cancer metastasis. G protein-coupled re- ceptor 137 （GPR137） was initially reported as a novel orphan GPR about 10 years ago. Some orphan GPRs have been implicated in human cancers. The aim of this study is to investigate the role of GPR137 in human colon cancer. Expression levels of GRP137 were analyzed in different colon cancer cell lines by quantitative polymerase chain re- action and western blot analysis. Lentivirus-mediated short hairpin RNA was specifically designed to knock down GPR137 expression in colon cancer cells. Cell viability was measured by methylthiazoletetrazolium and colony forma- tion assays. In addition, cell cycle characteristic was investi- gated by flow cytometry. GRP137 expression was observed in aH seven colon cancer cell lines at different levels. The mRNA and protein levels of GPR137 were down-regulated in both HCTll6 and RKO cells after lentivirus infection. Lentivirus-mediated silencing of GPR137 reduced the proliferation rate and colonies numbers. Knockdown of GPR137 in both cell lines led to cell cycle arrest in the G0/G1 phase. These results indicated that GPR137 plays an important role in colon cancer cell proliferation. A better understanding of GPR137＇s effects on signal transduction pathways in colon cancer cells may provide insights into the novel gene therapy of colon cancer.     

Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation

The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy.     

Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

Background

eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development.

Methods

We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis.

Results

Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues.

Conclusion

Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.