Pengxu Qian

Pivotal Role of Reduced let-7g Expression in Breast Cancer Invasion and Metastasis

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Gd-metallofullerenol nanomaterial as non-toxic breast cancer stem cell-specific inhibitor

The contemporary use of nanomedicines for cancer treatment has been largely limited to serving as carriers for existing therapeutic agents. Here, we provide definitive evidence that, the metallofullerenol nanomaterial Gd@C82(OH)22, while essentially not toxic to normal mammary epithelial cells, possesses intrinsic inhibitory activity against triple-negative breast cancer cells. Gd@C82(OH)22 blocks epithelial-to-mesenchymal transition with resultant efficient elimination of breast cancer stem cells (CSCs) resulting in abrogation of tumour initiation and metastasis. In normoxic conditions, Gd@C82(OH)22 mediates these effects by blocking TGF-β signalling. Moreover, under hypoxic conditions found in the tumour microenvironment, cellular uptake of Gd@C82(OH)22 is facilitated where it functions as a bi-potent inhibitor of HIF-1α and TGF-β activities, enhancing CSC elimination. These studies indicate that nanomaterials can be engineered to directly target CSCs. Thus, Gd-metallofullerenol is identified as a kind of non-toxic CSC specific inhibitors with significant therapeutic potential.

Loss of SNAIL Regulated miR-128-2 on Chromosome 3p22.3 Targets Multiple Stem Cell Factors to Promote Transformation of Mammary Epithelial Cells

A discontinuous pattern of LOH at chromosome 3p has been reported in 87% of primary breast cancers. Despite the identification of several tumor suppressor genes in this region, there has yet to be a detailed analysis of noncoding RNAs including miRNAs in this region. In this study, we identified 16 aberrant miRNAs in this region and determined several that are frequently lost or amplified in breast cancer. miR-128-2 was the most commonly deleted miRNA. Embedded in the intron of the ARPP21 gene at chromosome 3p22.3, miR-128-2 was frequently downregulated along with ARPP21 in breast cancer, where it was negatively associated with clinicopathologic characteristics and survival outcome. Forced expression of miR-128 impeded several oncogenic traits of mammary carcinoma cells, whereas depleting miR-128-2 expression was sufficient for oncogenic transformation and stem cell-like behaviors in immortalized nontumorigenic mammary epithelial cells, both in vitro and in vivo. miR-128-2 silencing enabled transforming capacity partly by derepressing a cohort of direct targets (BMI1, CSF1, KLF4, LIN28A, NANOG, and SNAIL), which together acted to stimulate the PI3K/AKT and STAT3 signaling pathways. We also found that miR-128-2 was directly downregulated by SNAIL and repressed by TGF-β signaling, adding 2 additional negative feedback loops to this network. In summary, we have identified a novel TGF-β/SNAIL/miR-128 axis that provides a new avenue to understand the basis for oncogenic transformation of mammary epithelial cells.

Tumor Expression of Human Growth Hormone and Human Prolactin Predict a Worse Survival Outcome in Patients with Mammary or Endometrial Carcinoma

CONTEXT Evidence suggests that human GH (hGH) and human prolactin (hPRL) possess an autocrine or paracrine oncogenic role in mammary and endometrial carcinoma. However, especially for hGH, the prognostic relevance of tumor expression of these hormones is not well defined. OBJECTIVE We investigated the potential association of tumor mRNA and protein expression of hGH and hPRL with the clinicopathological features of mammary and endometrial carcinoma. The prognostic relevance of the individual or combined expression of hGH and hPRL in mammary and endometrial carcinoma was also determined. DESIGN The expression of hGH and hPRL was analyzed in histopathological samples of mammary and endometrial carcinoma, and the respective normal tissues, by in situ hybridization and immunohistochemistry. Kaplan-Meier and Cox regression analysis was performed to examine the association of tumor hGH and hPRL expression with relapse-free survival and overall survival of patients. RESULTS hGH expression was significantly associated with lymph node metastasis, tumor stage, human epidermal growth factor receptor-2 status, and proliferative index in mammary carcinoma and with International Federation of Gynecology and Obstetrics grade, myometrial invasion, and ovarian metastases in endometrial carcinoma. hPRL expression was associated with lymph node metastasis, tumor grade, and tumor stage in mammary carcinoma and with International Federation of Gynecology and Obstetrics stage and myometrial invasion in endometrial carcinoma. Both hGH and hPRL expression, individually and combined, are associated with worse relapse-free survival and overall survival in patients with mammary or endometrial carcinoma. CONCLUSION Tumor expression of both hGH or hPRL in mammary or endometrial carcinoma is associated with a large and significant difference in survival outcome for patients with these tumors.

Artemin Stimulates Oncogenicity and Invasiveness of Human Endometrial Carcinoma Cells

Here, we provide evidence for a functional role of artemin (ARTN) in progression of endometrial carcinoma (EC). Increased ARTN protein expression was observed in EC compared with normal endometrial tissue, and ARTN protein expression in EC was significantly associated with higher tumor grade and invasiveness. Forced expression of ARTN in EC cells significantly increased total cell number as a result of enhanced cell cycle progression and cell survival. In addition, forced expression of ARTN significantly enhanced anchorage-independent growth and invasiveness of EC cells. Moreover, forced expression of ARTN increased tumor size in xenograft models and produced highly proliferative, poorly differentiated, and invasive tumors. The ARTN-stimulated increases in oncogenicity and invasion were mediated by increased expression and activity of AKT1. Small interfering RNA-mediated depletion or antibody inhibition of ARTN significantly reduced oncogenicity and invasion of EC cells. Thus, inhibition of ARTN may be considered as a potential therapeutic strategy to retard progression of EC.

Autocrine/Paracrine Human Growth Hormone-stimulated MicroRNA 96-182-183 Cluster Promotes Epithelial-Mesenchymal Transition and Invasion in Breast Cancer.

Background: hGH is an orthotopically expressed oncoprotein associated with mammary epithelial cell tumorigenesis. Results: The miR-96-182-183 cluster is regulated by autocrine/paracrine hGH and targets BRMS1L and GHR. Conclusion: Autocrine/paracrine hGH promotes breast cancer epithelial-mesenchymal transition and invasion via stimulating the miRNA-96-182-183 cluster. Significance: Autocrine/paracrine hGH and the miR-96-182-183 cluster might be exploited as a therapy or prognostic marker for breast cancer. Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development, and sexual maturation. Accumulated studies have reported that autocrine/paracrine hGH is an orthotopically expressed oncoprotein that promotes normal mammary epithelial cell oncogenic transformation. Autocrine/paracrine hGH has also been reported to promote mammary epithelial cell epithelial-mesenchymal transition (EMT) and invasion. However, the underlying mechanism remains largely obscure. MicroRNAs (miRNAs) are reported to be involved in regulation of multiple cellular functions of cancer. To determine whether autocrine/paracrine hGH promotes EMT and invasion through modulation of miRNA expression, we performed microarray profiling using MCF-7 cells stably expressing wild type or a translation-deficient hGH gene and identified miR-96-182-183 as an autocrine/paracrine hGH-regulated miRNA cluster. Forced expression of miR-96-182-183 conferred on epithelioid MCF-7 cells a mesenchymal phenotype and promoted invasive behavior in vitro and dissemination in vivo. Moreover, we observed that miR-96-182-183 promoted EMT and invasion by directly and simultaneously suppressing BRMS1L (breast cancer metastasis suppressor 1-like) gene expression. miR-96 and miR-182 also targeted GHR, providing a potential negative feedback loop in the hGH-GHR signaling pathway. We further demonstrated that autocrine/paracrine hGH stimulated miR-96-182-183 expression and facilitated EMT and invasion via STAT3 and STAT5 signaling. Consistent with elevated expression of autocrine/paracrine hGH in metastatic breast cancer tissue, miR-96-182-183 expression was also remarkably enhanced. Hence, we delineate the roles of the miRNA-96-182-183 cluster and elucidate a novel hGH-GHR-STAT3/STAT5-miR-96-182-183-BRMS1L-ZEB1/E47-EMT/invasion axis, which provides further understanding of the mechanism of autocrine/paracrine hGH-stimulated EMT and invasion in breast cancer.

ARTEMIN synergizes with TWIST1 to promote metastasis and poor survival outcome in patients with ER negative mammary carcinoma

IntroductionARTEMIN (ARTN) is an estrogen regulated growth factor, the expression of which promotes resistance to antiestrogen therapies and predicts poorer survival outcome of patients with estrogen receptor (ER) positive mammary carcinoma (ER+MC) treated with tamoxifen. ARTN is also expressed in ER negative mammary carcinoma (ER-MC). Herein, we determined the role of ARTN in ER-MC and defined the mechanism of action producing poor patient prognosis.MethodsWe modulated the expression of ARTN in two ER- (mesenchymal/claudin-low) mammary carcinoma cell lines (BT549 and MDA-MB-231) by forced expression or small interfering RNA (siRNA) mediated depletion. The effects of modulation of ARTN expression were examined by various in vitro measures of oncogenicity, including the expression of TWIST1 messenger RNA (mRNA) and protein. In vitro results were correlated to xenograft studies in immunodeficient mice. Co-expression of ARTN and TWIST1 and their association to poor survival outcome were examined in a cohort of patients with ER-MC. Pathway analysis was performed by pharmacological inhibition of phosphorylation of AKT (pAKT-Ser 473) or modulation of TWIST1 expression.ResultsARTN expression resulted in ER-MC cells with enhanced mesenchymal characteristics, including increased invasion and a gene expression profile consistent with enhanced mesenchymal phenotype. ARTN stimulated ER-MC cell anchorage independent and 3D matrigel growth, endothelial cell adhesion and transmigration of ER-MC cells through an endothelial cell barrier. Forced expression of ARTN produced a larger, locally invasive tumour mass with tumour emboli that produced distant metastasis. ARTN regulated TWIST1 expression in ER-MC cells and ARTN expression was significantly correlated to TWIST1 expression in a panel of mammary carcinoma cell lines and in a cohort of patients with ER-MC. Low expression of both ARTN and TWIST1 predicted 100% relapse free and overall survival in patients with ER-MC, whereas high expression of both ARTN and TWIST1 was associated with a poor survival outcome. ARTN stimulated an increase in TWIST1 expression via increased AKT activity. siRNA mediated depletion of TWIST1 abrogated ARTN stimulated cellular behaviour associated with metastasis, and forced expression of TWIST1 abrogated the functional effects of ARTN depletion.ConclusionsARTN and TWIST1 synergize to produce a worse outcome in ER-MC and combined inhibition of ARTN and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) may therefore provide a novel therapeutic strategy in this subtype of mammary carcinoma.

Artemin Stimulates Radio- and Chemo-resistance by Promoting TWIST1-BCL-2-dependent Cancer Stem Cell-like Behavior in Mammary Carcinoma Cells

Background: Artemin is an oncogenic and metastatic factor in mammary carcinoma. Results: Artemin promotes radio- and chemo-resistance by enhancing TWIST1-BCL-2-dependent cancer stem cell like behavior in mammary carcinoma cells. Conclusion: Artemin functions as a cancer stem cell (CSC) factor in mammary carcinoma cells. Significance: Functional inhibition of ARTN may be useful to inhibit CSC activity in mammary carcinoma. Artemin (ARTN) has been reported to promote a TWIST1-dependent epithelial to mesenchymal transition of estrogen receptor negative mammary carcinoma (ER-MC) cells associated with metastasis and poor survival outcome. We therefore examined a potential role of ARTN in the promotion of the cancer stem cell (CSC)-like phenotype in mammary carcinoma cells. Acquired resistance of ER-MC cells to either ionizing radiation (IR) or paclitaxel was accompanied by increased ARTN expression. Small interfering RNA (siRNA)-mediated depletion of ARTN in either IR- or paclitaxel-resistant ER-MC cells restored cell sensitivity to IR or paclitaxel. Expression of ARTN was enriched in ER-MC cells grown in mammospheric compared with monolayer culture and was also enriched along with BMI1, TWIST1, and DVL1 in mammospheric and ALDH1+ populations. ARTN promoted mammospheric growth and self-renewal of ER-MC cells and increased the ALDH1+ population, whereas siRNA-mediated depletion of ARTN diminished these CSC-like cell behaviors. Furthermore, increased ARTN expression was significantly correlated with ALDH1 expression in a cohort of ER-MC patients. Forced expression of ARTN also dramatically enhanced tumor initiating capacity of ER-MC cells in xenograft models at low inoculum. ARTN promotion of the CSC-like cell phenotype was mediated by TWIST1 regulation of BCL-2 expression. ARTN also enhanced mammosphere formation and the ALDH1+ population in estrogen receptor-positive mammary carcinoma (ER+MC) cells. Increased expression of ARTN and the functional consequences thereof may be one common adaptive mechanism used by mammary carcinoma cells to promote cell survival and renewal in hostile tumor microenvironments.

ARTEMIN Promotes De Novo Angiogenesis in ER Negative Mammary Carcinoma through Activation of TWIST1-VEGF-A Signalling

The neurotrophic factor ARTEMIN (ARTN) has been reported to possess a role in mammary carcinoma progression and metastasis. Herein, we report that ARTN modulates endothelial cell behaviour and promotes angiogenesis in ER-mammary carcinoma (ER-MC). Human microvascular endothelial cells (HMEC-1) do not express ARTN but respond to exogenously added, and paracrine ARTN secreted by ER-MC cells. ARTN promoted endothelial cell proliferation, migration, invasion and 3D matrigel tube formation. Angiogenic behaviour promoted by ARTN secreted by ER-MC cells was mediated by AKT with resultant increased TWIST1 and subsequently VEGF-A expression. In a patient cohort of ER-MC, ARTN positively correlated with VEGF-A expression as measured by Spearman’s rank correlation analysis. In xenograft experiments, ER-MC cells with forced expression of ARTN produced tumors with increased VEGF-A expression and increased microvessel density (CD31 and CD34) compared to tumors formed by control cells. Functional inhibition of ARTN by siRNA decreased the angiogenic effects of ER-MC cells. Bevacizumab (a humanized monoclonal anti-VEGF-A antibody) partially inhibited the ARTN mediated angiogenic effects of ER-MC cells and combined inhibition of ARTN and VEGF-A by the same resulted in further significant decrease in the angiogenic effects of ER-MC cells. Thus, ARTN stimulates de novo tumor angiogenesis mediated in part by VEGF-A. ARTN therefore co-ordinately regulates multiple aspects of tumor growth and metastasis.

CCAR1 5′ UTR as a natural miRancer of miR-1254 overrides tamoxifen resistance

MicroRNAs (miRNAs) typically bind to unstructured miRNA-binding sites in target RNAs, leading to a mutual repression of expression. Here, we report that miR-1254 interacts with structured elements in cell cycle and apoptosis regulator 1 (CCAR1) 5′ untranslated region (UTR) and this interaction enhances the stability of both molecules. miR-1254 can also act as a repressor when binding to unstructured sites in its targets. Interestingly, structured miR-1254-targeting sites act as both a functional RNA motif-sensing unit, and an independent RNA functional unit that enhances miR-1254 expression. Artificially designed miRNA enhancers, termed “miRancers”, can stabilize and enhance the activity of miRNAs of interest. We further demonstrate that CCAR1 5′ UTR as a natural miRancer of endogenous miR-1254 re-sensitizes tamoxifen-resistant breast cancer cells to tamoxifen. Thus, our study presents a novel model of miRNA function, wherein highly structured miRancer-like motif-containing RNA fragments or miRancer molecules specifically interact with miRNAs, leading to reciprocal stabilization.