Ruey-Jen Lin

miR-149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells

microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been widely explored. However, it remains to be ascertained whether Akt1, the predominant isoform in most tissue, is a direct target of miRNA. In this study, we identified Akt1 and E2F1 to be two direct targets of miR‐149* and b‐Myb to be an indirect target by reporter assays and Western blot analyses. Ectopic expression of miR‐149*‐induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro‐apoptotic effects of miR‐149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR‐149* with E2F1 expressions (P = 0.026). Interestingly, using the reporter assays, excess miR‐149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR‐149* on the target genes. This implies that the pro‐apoptotic function of miR‐149* may not be dampened by its predominant cognate, miR‐149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR‐149* is a pro‐apoptotic miRNA by repressing the expression of Akt1 and E2F1.

c-Myb Is an Evolutionary Conserved miR-150 Target and miR-150/c-Myb Interaction Is Important for Embryonic Development

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.

The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

IntroductionDysregulation of the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway was shown to correlate with breast cancer disease progression. Cancer stem cells are a subpopulation within cancer cells that participate in tumor initiation, radio/chemoresistance and metastasis. In breast cancer, breast cancer stem cells (BCSCs) were identified as CD24-CD44+ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Elucidation of the role of IGF-1R in BCSCs is crucial to the design of breast cancer therapies targeting BCSCs.MethodsIGF-1R expression in BCSCs and noncancer stem cells sorted from xenografts of human primary breast cancers was examined by fluorescence-activated cell sorting (FACS), western blot analysis and immunoprecipitation. The role of IGF-1R in BCSCs was assessed by IGF-1R blockade with chemical inhibitor and gene silencing. Involvement of PI3K/Akt/mammalian target of rapamycin (mTOR) as the downstream pathway was studied by their phosphorylation status upon IGF-1R inhibition and the effects of chemical inhibitors of these signaling molecules on BCSCs. We also studied 16 clinical specimens of breast cancer for the expression of phosphor-Akt in the BCSCs by FACS.ResultsExpression of phosphorylated IGF-1R was greater in BCSCs than in non-BCSCs from xenografts of human breast cancer, which were supported by western blot and immunoprecipitation experiments. The sorted IGF-1R-expressing cells displayed features of cancer stem/progenitors such as mammosphere formation in vitro and tumorigenicity in vivo, both of which were suppressed by knockdown of IGF-1R. A specific inhibitor of the IGF-1R, picropodophyllin suppressed phospho-AktSer473 and preferentially decreased ALDH+ BCSC populations of human breast cancer cells. Furthermore, picropodophyllin inhibited the capacity of CD24-CD44+ BCSCs to undergo the epithelial-mesenchymal transition process with downregulation of mesenchymal markers. Inhibitors of signal molecules downstream of IGF-1R including PI3K/Akt/mTOR also reduced the ALDH+ population of breast cancer cells. Furthermore, the mTOR inhibitor, rapamycin, suppressed BCSCs in vitro and in vivo.ConclusionOur data support the notion that IGF-1R is a marker of stemness, and IGF-1R and its downstream PI3K/Akt/mTOR pathway are attractive targets for therapy directed against breast cancer stem/progenitors.

Concordant and Discordant Regulation of Target Genes by miR-31 and Its Isoforms

It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA) variants designated as “isomiR”. Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M), which differ only slightly in their 5′- and/or 3′-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3′-untranslated region (UTR) of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

A Novel Oncogenic Role of Inositol Phosphatase SHIP2 in ER‐Negative Breast Cancer Stem Cells: Involvement of JNK/Vimentin Activation

Overexpression of SH2‐containing‐5′‐inositol phosphatase‐2 (SHIP2) correlates with poor survival in breast cancer. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here, we showed that the percentage of SHIP2+ cells was positively correlated with that of CD24−CD44+ cells in 60 breast cancer specimens. Among 20 estrogen receptor (ER)‐negative samples, 17 had greater SHIP2 expression in CD24−CD44+ subpopulation than the remaining subpopulation. Data mining of microarray analysis of 295 breast tumors showed a significant correlation of higher SHIP2 expression with distant metastasis. Examination of patient‐derived mouse xenografts revealed that SHIP2 protein and its tyrosine 1135 phosphorylation were significantly higher in BCSCs, identified as CD24−CD44+ or aldehyde dehydrogenase (ALDH+), than non‐BCSCs. SHIP2 silencing or inhibitor of SHIP2 phosphatase significantly decreased mammosphere‐forming efficiency, ALDH+ subpopulation in vitro and tumorigenicity of BCSCs in vivo. Overexpression of SHIP2 enhanced the expression of epithelial–mesenchymal transition markers including vimentin (VIM), which was mainly expressed in ER‐negative breast cancer cells with higher level in mammospheres than monolayer culture. Ablation of c‐Jun N‐terminal kinase 1 (JNK1), JNK2, or VIM diminished the increased ALDH+ population and tumorigenicity, induced by SHIP2 overexpression. BCSCs displayed greater expression of phospho‐JNK than non‐BCSCs and silencing of JNK suppressed SHIP2‐mediated upregulation of VIM. Furthermore, SHIP2 overexpression enhanced Akt activation, but Akt inhibition failed to influence SHIP2‐induced phospho‐JNK/VIM upregulation. In conclusion, SHIP2 plays a key role in BCSCs of ER‐negative breast cancers through activation of Akt and JNK with upregulation of VIM and may serve as a target for therapy directed at BCSCs. Stem Cells 2014;32:2048–2060

Abstract 980: The long non-coding RNA CAI2 is a tumor suppressor gene

We have recently reported on the discovery of a long non-coding RNA imbedded in intron 2 of the p16/ARF (CDKN2A) locus at 9p21. CAI2 is a non-conserved, RNA pol II regulated gene with whose expression is highly correlated with parent genes p16 and ARF. However, CAI2 is expressed in cell lines deleted for p16, ARF and even p15, and is expressed cell lines epigenetically silenced for p16 and/or ARF. Furthermore, treatment of p16 and/or ARF epigenetically silenced cell lines with 5-aza-2’-deoxycytidin allows p16 and/or ARF re-expression while having no influence on CAI2, confirming independent regulation. Speculating, therefore, that the regulatory region of CAI2 lies imbedded in the non-transcribed portion of intron 2, we created expression constructs of the immediate 5’ region of CAI2. However, consistent with a bioinformatical analysis, no regulatory activity was detected. This suggests that this non-coding RNA is non-traditionally controlled by remote but yet to be identified regulatory element(s) and/or mechanisms. By colony formation assay, CAI2 overexpression can inhibit cell growth in the p16 intact HEK293T and SK-BR-3 breast cancer cell lines. RNA Immunoprecipitation has revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression, and BMI1, a PRC1 complex protein that is a negative regulator of p16. Consistent with this effect, CAI2 can induce p16 expression in HEK293T cells, and this was hypothesized to be the mechanism by which CAI2 inhibits cell growth. However, we now show that CAI2 can also inhibit colony formation in the p16/ARF deleted MCF7 and MDA-MB-231 breast cancer cell lines. Interestingly however, CAI2 overexpression had little to no effect on 2D cultures of HEK293T or MCF7 cells including DNA synthesis, XTT metabolic activity or cell count. As CAI2 is imbedded in the p16/ARF gene, standard siRNAs may inadvertently influence p16 and/or ARF expression due to actions on their pre-mRNAs. Therefore, we created a series of partial CAI2 knockouts using CRISPR technology and compared their growth by colony formation assay. Notably, clones containing a partial knockout of the 5’ region of CAI2 proliferated faster than mock transfected clones, consistent with the growth suppressive properties observed with CAI2 overexpression. In 2D cultures, a small increase in DNA synthesis was also observed in the partial deletions. The HEK293T clones containing partial deletions of CAI2 also exhibited small increases in expression of the remaining intact portions of the gene with a concomitant decrease in p16 expression, all consistent with an influence of CAI2 on p16 expression and cell growth; no influence on ARF expression was observed. Taken together, the data demonstrate that CAI2 is capable of influencing p16 expression, but this influence is insufficient to fully account for the ability of CAI2 to regulate growth, and we conclude that CAI2 is a bona fide tumor suppressor gene in its own right. Citation Format: Olga Cohen, Ruey-Jen Lin, Alice L. Yu, Mitchell B. Diccianni. The long non-coding RNA CAI2 is a tumor suppressor gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 980.

Abstract 5197: Quantitative phosphorproteomics identified GPER-initiated PKA signal transduction event in breast cancer stem cell

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Human breast cancer stem cells (BCSC) identified recently have the ability to initiate neoplastic growth with differentiation and display higher resistance to chemotherapy and radiotherapy. The allure of targeting this specific group of cells to reduce mortality of disease has attracted research efforts toward BCSC. Although some unique properties of BCSC have been deciphered, the molecular mechanisms are still poorly understood. Here, we applied in-house developed techniques to quantitatively compare the phosphoproteomic profiles between BCSC and non-cancer stem cells (nonCSC) derived from a xenograft of human breast cancer. For quantitative analysis, we utilized a SEMI-strategy for multiplexed label-free quantitation combining pH/acid-controlled IMAC chromatography and LC-MS/MS for relative quantitation of site-specific phosphorylation. This strategy effectively increases the phosphopeptide quantitation coverage. The differentially expressed phosphoproteins were mapped to multiple signaling pathways including NOTCH, CDK/ERK and JAK-STAT pathways, which potentially orchestrate the self renewal and stemness of BCSC. We also demonstrated ∼2 fold greater expression of GPER in BCSC than non-BCSC population, and GPER signaling could induce PKA-mediated phosphorylation of BAD in BCSC. Silencing of GPER via RNAi or mutation of phosphorylation sites of BAD led to reduced proliferation to 40.8 and 53.2 % of control, respectively, and the RNAi silencing of GPER decreased the mammosphere formation of BCSC to 43.5 % of control. These findings implies the importance of GPER and its downstream PKA pathway to the maintenance of stemness characteristics of BCSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5197. doi:1538-7445.AM2012-5197

Abstract 3983: MiR-31 isoforms differentially regulate expression of dicer in cancer cells

MicroRNAs (miRNAs) are a group of small non-coding RNAs involved in several important biological processes. Recent studies have implied that imprecise cleavage of primary or precursor RNA by Drosha or Dicer respectively could produce miRNAs with variations at 5’-and/or 3’-ends, which were termed “isomiR”. The existence of isomiRs may increase the complexity of miRNA biology, although their functions have not been explored. This is further complicated by the observation of variations in the population of isomer for a given miRNA among different species as well as among different types of cells within the same species (human cells), which raises the issue whether these isomers might exert differential effects on target gene regulation. Here, we utilized three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M) which differ from each other only slightly in their sequences at both ends, to compare their capacity to regulate a predicted target, Dicer. Interestingly, we found only miR-31-P was able to directly repress Dicer expression in both breast cancer MCF-7 cells and lung cancer A549 cells, resulting in their enhanced sensitivity to cisplatin, which is known to be associated with Dicer knockdown. In line with this, the amount of miR-31-P bound to the RNA-induced silencing complex (RISC) was greater than that of miR-31-H and -M by RNA-CHIP analysis. Furthermore, evaluation of other known miR-31 target genes showed that expression of some target genes was indeed differentially regulated by miR-31 isoforms. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated the disparate functions of isomiRs for the fist time, providing a novel functional mechanism of fine-tuning of gene expression by miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2011-3983