Peng Lee

Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor.

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31–34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182s proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy.

A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas

Human uterine leiomyomas (ULMs) are the most common neoplasms of women. Many genes are dysregulated in ULMs and some of this dysregulation may be due to abnormal expression of micro‐RNAs (miRNAs). In this study, 55 ULMs and matched myometrium were collected from 41 patients for microarray‐based global miRNA expression analysis. Of 206 miRNAs examined, 45 miRNAs were significantly up‐ or down‐regulated in ULMs in comparison to the matched myometrium (P < 0.001). The top five dysregulated miRNAs in ULMs are the let‐7 family, miR‐21, miR‐23b, miR‐29b, and miR‐197. Four polycistronic clusters of miRNAs were either up‐ or down‐regulated, but not in a mixed pattern, indicative of coordinated regulation of these miRNAs. Significance analysis revealed that subsets of miRNAs were strongly associated with tumor sizes and race. By prediction analysis we identified some important tumorigenic genes previously identified in ULMs that may be targeted by the dysregulated miRNAs. HMGA2 was identified as one of target genes of the let‐7 family of miRNAs and has been found to be suppressed by let‐7 in vitro. (This article contains Supplementary material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.)

Antiproliferative effects by Let-7 repression of high-mobility group A2 in uterine leiomyoma.

High-mobility group A2 (HMGA2) is commonly overexpressed in large leiomyomas. HMGA2 is an important regulator of cell growth, differentiation, apoptosis, and transformation. As a predicted target of Let-7 microRNAs (Let-7s), HMGA2 can be repressed by Let-7s in vitro. MicroRNA profiling analysis revealed that Let-7s were significantly dysregulated in uterine leiomyomas: high in small leiomyomas and lower in large leiomyomas. To evaluate whether Let-7 repression of HMGA2 plays a major role in leiomyomas, we analyzed the molecular relationship of HMGA2 and Let-7s, both in vitro and in vivo. We first characterized that exogenous Let-7 microRNAs could directly repress the dominant transcript of HMGA2, HMGA2a. This repression was also identified for two cryptic HMGA2 transcripts in primary leiomyoma cultures. Second, we found that the endogenous Let-7s were biologically active and played a major role in the regulation of HMGA2. Then, we illustrated that Let-7 repression of HMGA2 inhibited cellular proliferation. Finally, we examined the expression levels of Let-7c and HMGA2 in a large cohort of leiomyomas (n = 120), and we found high levels of Let-7 and low levels of HMGA2 in small leiomyomas, and low levels of Let-7 and high levels of HMGA2 in large leiomyomas. Our findings suggest that the Let-7–mediated repression of HMGA2 mechanism can be an important molecular event in leiomyoma growth. (Mol Cancer Res 2008;6(4):663–73)

Cell-specific Regulation of Androgen Receptor Phosphorylation in Vivo

The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu.

HMGA2 overexpression-induced ovarian surface epithelial transformation is mediated through regulation of EMT genes.

The AT-hook transcription factor HMGA2 is an oncogene involved in the tumorigenesis of many malignant neoplasms. HMGA2 overexpression is common in both early and late-stage high-grade ovarian serous papillary carcinoma. To test whether HMGA2 participates in the initiation of ovarian cancer and promotion of aggressive tumor growth, we examined the oncogenic properties of HMGA2 in ovarian surface epithelial (OSE) cell lines. We found that introduction of HMGA2 overexpression was sufficient to induce OSE transformation in vitro. HMGA2-mediated OSE transformation resulted in tumor formation in the xenografts of nude mice. By silencing HMGA2 in HMGA2-overexpressing OSE and ovarian cancer cell lines, the aggressiveness of tumor cell growth behaviors was partially suppressed. Global gene profiling analyses revealed that HMGA2-mediated tumorigenesis was associated with expression changes of target genes and microRNAs that are involved in epithelial-to-mesenchymal transition (EMT). Lumican, a tumor suppressor that inhibits EMT, was found to be transcriptionally repressed by HMGA2 and was frequently lost in human high-grade serous papillary carcinoma. Our findings show that HMGA2 overexpression confers a powerful oncogenic signal in ovarian cancers through the modulation of EMT genes.

MiR‐182 overexpression in tumourigenesis of high‐grade serous ovarian carcinoma

Molecular pathogenesis of high‐grade serous ovarian carcinoma (HG‐SOC) is poorly understood. Recent recognition of HG‐SOC precursor lesions, defined as serous tubal intraepithelial carcinoma (STIC) in fimbria, provides a new venue for the study of early genetic changes in HG‐SOC. Using microRNA profiling analysis, we found that miR‐182 expression was significantly higher in STIC than in matched normal Fallopian tube. Further study revealed that miR‐182 was significantly overexpressed in most HG‐SOC cases. To test whether miR‐182 plays a major role in early tumourigenesis of HG‐SOC, we overexpressed miR‐182 in immortalized ovarian surface, Fallopian tube secretory cells and malignant ovarian cell lines, and found that miR‐182 overexpression resulted in increased tumour transformation in vitro, and enhanced tumour invasiveness in vitro and metastasis in vivo. Mechanistically, we demonstrated that the oncogenic properties of miR‐182 in ovarian cancer were mediated in part by its impaired repair of DNA double‐strand breaks and negative regulation of breast cancer 1 (BRCA1) and metastasis suppressor 1 (MTSS1) expression as well as its positive regulation of the oncogene high‐mobility group AT‐hook 2 (HMGA2). Our findings suggest that miR‐182 dysregulation confers powerful oncogenic potential in the tumourigenesis of HG‐SOC. Copyright

Racial Disparity of Epidermal Growth Factor Receptor Expression in Prostate Cancer

PURPOSE The epidermal growth factor receptor (EGFR) plays a critical role in prostate cancer (PC) signal transduction and is the target of a novel class of anticancer agents. Despite recent reports of interethnic variation in response to EGFR inhibitors, limited information exists regarding differences in expression of EGFR in PC patients. This has therapeutic relevance because a better understanding of the molecular basis underlying the ethnic variability will help in the design of individualized treatment regimens using EGFR inhibitors. PATIENTS AND METHODS We investigated EGFR expression in a well-characterized cohort of PC patients to determine the association between EGFR expression and race. Tumor tissues from 202 radical prostatectomies performed between 1990 and 2000 at the Veterans Administration Medical Center (New York, NY) were studied (142 African Americans, 60 whites; median age, 67 years; stage T2, n = 130; stage > or = T3, n = 72; Gleason score < 7, n = 110; Gleason score > or = 7, n = 92). Membrane-specific EGFR expression was evaluated immunohistochemically. RESULTS EGFR overexpression, defined as complete membrane staining in more than 10% of tumor cells, was observed in 75 of 202 patients (37%). There was a significant association between EGFR overexpression and African American race (P = .0006), higher pretreatment prostate-specific antigen (PSA; P = .02), and stage (P = .02), but not Gleason score (P = .33). The association between African American race and EGFR overexpression remained significant in a multivariate model after controlling for grade, stage, and pretreatment PSA simultaneously (P = .003). CONCLUSION Our data demonstrate that race contributes significantly to variability of EGFR expression in prostate cancer. Racial background may have an impact on the design of clinical trials to test the efficacy of anti-EGFR agents.

Androgen receptor splice variants dimerize to transactivate target genes

Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR(v567es), not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer.

Regulation of HMGA1 Expression by MicroRNA-296 Affects Prostate Cancer Growth and Invasion

Purpose: High-motility group AT-hook gene 1 (HMGA1) is a non-histone nuclear binding protein that is developmentally regulated. HMGA1 is significantly overexpressed in and associated with high grade and advance stage of prostate cancer (PC). The oncogenic role of HMGA1 is at least mediated through chromosomal instability and structural aberrations. However, regulation of HMGA1 expression is not well understood. Identification of microRNA-mediated HMGA1 regulation will provide a promising therapeutic target in treating PC. Experimental Design: In this study, we examined the functional relation between miR-296 and HMGA1 expression in several PC cell lines and a large PC cohort. We further examined the oncogenic property of HMGA1 regulated by miR-296. Results: Here we report that miR-296, a microRNA predicted to target HMGA1, specifically represses HMGA1 expression by promoting degradation and inhibiting HMGA1translation. Repression of HMGA1 by miR-296 is direct and sequence specific. Importantly, ectopic miR-296 expression significantly reduced PC cell proliferation and invasion, in part through the downregulation of HMGA1. Examining PC patient samples, we found an inverse correlation between HMGA1 and miR-296 expression: high levels of HMGA1 were associated with low miR-296 expression and strongly linked to more advanced tumor grade and stage. Conclusions: Our results indicate that miR-296 regulates HMGA1 expression and is associated with PC growth and invasion. Clin Cancer Res; 17(6); 1297–305. ©2010 AACR.

LEF1 in Androgen-Independent Prostate Cancer: Regulation of Androgen Receptor Expression, Prostate Cancer Growth, and Invasion

A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease.