The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.     

Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling

Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P<0.01). DACH1 methylation was associated with poor differentiation (P<0.05) and late tumor stage (P<0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-β signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-β signaling.     

Xanthohumol Impairs Human Prostate Cancer Cell Growth and Invasion and Diminishes the Incidence and Progression of Advanced Tumors in TRAMP Mice

Despite recent advances in understanding the biological basis of prostate cancer, management of the disease, especially in the phase resistant to androgen ablation, remains a significant challenge. The long latency and high incidence of prostate carcinogenesis provides the opportunity to intervene with chemoprevention to prevent or eradicate prostate malignancies. In this study, we have used human hormone-resistant prostate cancer cells, DU145 and PC3, as an in vitro model to assess the efficacy of xanthohumol (XN) against cell growth, motility and invasion. We observed that treatment of prostate cancer cells with low micromolar doses of XN inhibits proliferation and modulates focal adhesion kinase (FAK) and AKT phosphorylation leading to reduced cell migration and invasion. Oxidative stress by increased production of reactive oxygen species (ROS) was associated with these effects. Transgenic adenocarcinoma of the mouse prostate (TRAMP) transgenic mice were used as an in vivo model of prostate adenocarcinoma. Oral gavage of XN, three times per week, beginning at 4 wks of age, induced a decrease in the average weight of the urogenital (UG) tract, delayed advanced tumor progression and inhibited the growth of poorly differentiated prostate carcinoma. The ability of XN to inhibit prostate cancer in vitro and in vivo suggests that XN may be a novel agent for the management of prostate cancer.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

Exploration of Shh and BMP paracrine signaling in a prostate cancer xenograft

Stromal–epithelial signaling is a critical regulator of normal prostate development and has been speculated to play an equally important role in the development and progression of prostate cancer. Sonic hedgehog (Shh) and bone morphogenetic proteins (BMP-4, BMP-7), expressed by the urogenital sinus epithelium and mesenchyme, exert reciprocal and coordinate effects on outgrowth of nascent prostate ducts. Over-expression of Shh in the LNCaP xenograft was shown previously to accelerate tumor growth by a paracrine mechanism. A survey of BMP regulators expressed in the developing prostate revealed increased Noggin and BMP-7 mRNA in the stromal component of Shh over-expressing xenografts. In vitro studies demonstrated that treatment of LNCaP cells with BMP-4 and BMP-s7 induced Id-1 expression and inhibited tumor cell proliferation. The activity of BMP-4 was abrogated by co-addition of Noggin; the activity of BMP-7 was not. Quantitative analysis of BMP signaling revealed ambivalent results: decreased tumor cell expression of the BMP response gene Id-1 but increased staining for phospho-SMAD 1,5, 8. To directly test whether increased xenograft tumor growth could be explained by Noggin-mediated blockade of BMP-2/4 effects on tumor cell proliferation, we generated LNCaP xenografts containing stromal cells over-expressing Noggin. Tumor cells in these xenografts exhibited decreased Id-1 and reduced SMAD phosphorylation, but tumor growth was not altered. We conclude that tumor cell Shh expression can induce significant changes in expression of BMP ligands and inhibitors in the stromal microenvironment but that acceleration of LNCaP xenograft tumor growth by Shh over-expression cannot be attributed solely to increased Noggin expression in the tumor stroma.     

Restoration of cyclin D2 has an inhibitory potential on the proliferation of LNCaP cells

Despite well known oncogenic function of G1-S cell-cycle progression, cyclin D2 (CCND2) is often silenced epigenetically in prostate cancers. Here we show that CCND2 has an inhibitory potential on the proliferation of androgen receptor (AR)-dependent prostate cancer LNCaP cells. Forced expression of CCND2 suppressed the proliferative ability and induced cell death in LNCaP cells in a cdk-independent manner. Knocking down CCND2 restored the proliferation of LNCaP subclones with relatively high CCND2 expression and low proliferative profiles. Immunoprecipitation using deletion mutants of CCND2 indicated that a central domain of CCND2 is required for binding to AR. A deletion mutant lacking the central domain failed to hinder LNCaP cells. Collectively, our results indicated that CCND2 inhibits cell proliferation of AR-dependent prostate cancer through the interaction with AR. Our study suggests that restoration of CCND2 expression potentially prevents the carcinogenesis of prostate cancer, which is mostly AR-dependent in the initial settings.     

In Vivo Targeting of ADAM9 Gene Expression Using Lentivirus-Delivered shRNA Suppresses Prostate Cancer Growth by Regulating REG4 Dependent Cell Cycle Progression

Cancer cells respond to stress by activating a variety of survival signaling pathways. A disintegrin and metalloproteinase (ADAM) 9 is upregulated during cancer progression and hormone therapy, functioning in part through an increase in reactive oxygen species. Here, we present in vitro and in vivo evidence that therapeutic targeting of ADAM9 gene expression by lentivirus-delivered small hairpin RNA (shRNA) significantly inhibited proliferation of human prostate cancer cell lines and blocked tumor growth in a murine model of prostate cancer bone metastasis. Cell cycle studies confirmed an increase in the G1-phase and decrease in the S-phase population of cancer cells under starvation stress conditions, which correlated with elevated intracellular superoxide levels. Microarray data showed significantly decreased levels of regenerating islet-derived family member 4 (REG4) expression in prostate cancer cells with knockdown of ADAM9 gene expression. This REG4 downregulation also resulted in induction of expression of p21^Cip1/WAF1, which negatively regulates cyclin D1 and blocks the G1/S transition. Our data reveal a novel molecular mechanism of ADAM9 in the regulation of prostate cancer cell proliferation, and suggests a combined modality of ADAM9 shRNA gene therapy and cytotoxic agents for hormone refractory and bone metastatic prostate cancer.     

Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer

IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO– terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis.     

HOXD8 inhibits the proliferation and migration of triple-negative breast cancer cells and induces apoptosis in them through regulation of AKT/mTOR pathway

HOXD8 (Homeobox D8) functions as an apoptotic inducer to suppress tumor progression. However, the role of HOXD8 in triple-negative breast cancer (TNBC) has not been fully understood. Firstly, HOXD8 was found to be reduced in TNBC tissues based on the TCGA samples through Ualcan (http://ualcan.path.uab.edu/analysis.html) prediction. Moreover, data from qRT-PCR and western blot confirmed the lower expression of HOXD8 in the TNBC tissues or cells than that in paracancerous tissues or human mammary epithelial cell line (MCF10A), respectively. Secondly, pcDNA-mediated over-expression of HOXD8 were conducted in TNBC cells, and the gain-of functional assays showed that over-expression of HOXD8 promoted TNBC cell progression with repressed cell apoptosis and induced proliferation, migration and invasion. Moreover, xenografted mouse model was constructed by injection of tumor cell line with stable over-expression of HOXD8 to assess the in vivo tumor growth, and the results revealed that over-expression of HOXD8 inhibited tumor growth. Lastly, our results showed that AKT and mTOR phosphorylation were repressed by HOXD8 over-expression in TNBC cells. In conclusion, HOXD8 functioned as an apoptotic inducer to suppress TNBC cell growth and progression by inhibition of AKT/mTOR pathway.