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Ethnicity is considered to be one of the major risk factors in certain subtypes of breast cancer. However, the mechanism of this racial disparity remains poorly understood. Here, we demonstrate that SOS1, a key regulator of Ras pathway, is highly expressed in African-American (AA) patients with breast cancer compared with Caucasian-American patients. Because of the higher obesity rate in AA women, increased levels of SOS1 facilitated signal transduction of the c-Met pathway, which was highly activated in AA patients with breast cancer via hepatocyte growth factor secreted from adipocytes. Elevated expression of SOS1 also enhanced cancer stemness through upregulation of PTTG1 and promoted M2 polarization of macrophages by CCL2 in metastatic sites. SOS1 was epigenetically regulated by a super-enhancer identified by H3K27ac in AA patients. Knockout of the super-enhancer by CRISPR in AA cell lines significantly reduced SOS1 expression. Furthermore, SOS1 was posttranscriptionally regulated by miR-483 whose expression is reduced in AA patients through histone trimethylation (H3K27me3) on its promoter. The natural compound, taxifolin, suppressed signaling transduction of SOS1 by blocking the interaction between SOS1 and Grb2, suggesting a potential utility of this compound as a therapeutic agent for AA patients with breast cancer. Significance: These findings elucidate the signaling network of SOS1-mediated metastasis in African-American patients, from the epigenetic upregulation of SOS1 to the identification of taxifolin as a potential therapeutic strategy against SOS1-driven tumor progression.

Background Few currently available biomarker panels can predict the risk of long-term distant recurrence (DR) in endocrine-responsive breast cancer (ERBC) patients based on lymph-node (LN) status. A new model, DGM-CM6 (Distant Genetic Model-Clinical variable Model 6), has been developed for the prediction of DR risk in our previous studies. To interrogate the prognostic value of our DGM-CM6 model for ERBC patients stratified by LN status, we retrospectively studied 752 operable breast cancer patients treated in a cancer center from 2005 to 2014. Patients and Methods Of 752 tumors, a total of 499 ERBC patients was identified from both IHC method (n=490) and PAM50 method (n=404). ERBC patients were classified by high and low-risk groups using the cutoff ( Results Significant differences were observed between DGM-CM6 low- and high-risk patients with 10-year DRFS in LN- (ERBC identified by IHC: P = 0.011; by PAM50: P< 0.0001) and LN+ (ERBC identified by IHC: P = 0.015; by PAM50: P = 0.019) by log-rank test. Compared to risk prediction by intrinsic subtype, DGM-CM6 performed better in LN- patients and equally good in LN+ patients. Further multivariate analysis confirmed the independent strength of DGM-CM6 model in the prediction of high- vs. low- risk groups in DRFS (P < 0.0001, HR: 6.76; 95% CI, 1.8-25.42) and OS (P = 0.01, HR: 6.06; 95% CI:1.55-23.47), respectively. Conclusion DGM-CM6 model can be used to predict low- and high-risk of 10-year distant recurrence in both LN- and LN+ ERBC patients. This model needs a large scale of clinical trials to validate its clinical utility. Table 1. Multivariate cox regression analyses for the prognosis of predicted risk groups after adjusted for other clinical variables in the distant recurrence (DR, p=0.005) and overall survival (OS, p=0.01) respectively. Results shows that there is no interaction between chemotherapy and risk groups (DR: p=0.163; OS: p=0.195). Abbreviations: DR, distant recurrence; OS: overall survival.GroupsDROSHR [95% CI]pvHR [95% CI]pvRisk: high vs.low6.76 [1.8;25.42]0.005 ***6.06 [1.55;23.74]0.01 ***Age: 50yrs0.86 [0.51;1.44]0.5630.68 [0.38;1.19]0.175LN: Pos. vs. Neg.1.64 [0.88;3.05]0.1161.6 [0.79;3.22]0.189Stage: II vs. I1.35 [0.76;2.4]0.31.45 [0.76;2.77]0.264Stage: III vs. I2.08 [0.59;7.3]0.2531.59 [0.34;7.46]0.557Grade: 2 vs. 12.14 [0.77;5.97]0.1461.81 [0.63;5.25]0.272Grade: 3 vs. 11.2 [0.4;3.62]0.7481.21 [0.38;3.86]0.752PAM50: Normal vs. LumA0.6 [0.13;2.64]0.4960.7 [0.16;3.16]0.645PAM50: LumB vs. LumA1.58 [0.84;2.98]0.1591.29 [0.65;2.59]0.469PAM50: Her2 vs. LumA0.81 [0.25;2.68]0.7311.01 [0.29;3.47]0.986RT: Yes vs. No0.88 [0.47;1.66]0.7010.71 [0.36;1.41]0.326CT: Yes vs.No0.36 [0.11;1.12]0.0770.3 [0.09;1]0.05 *Interaction: CT vs. Risk0.36 [0.09;1.51]0.1630.36 [0.08;1.68]0.195 Citation Format: Lei Lei, Skye Hung-Chun Cheng, Xiao-Jia Wang, Yin-Yuan Mo, Yun-Yun Zhou. A new model to predict 10-year distant recurrence risk for operable endocrine-responsive breast cancer population [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-14.

Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.

N6-methyladenosine (m6A) is one of the most prevalent modifications of mRNAs in eukaryotes. Numerous recent studies have indicated that m6A plays the critical role in multiple physiologic functions and disease processes by regulation of mRNA processing, splicing, translation, and stability. Although the majority of RNA methylation studies focus on mRNAs, little is known whether long-coding RNAs (lncRNAs) are also subject to m6A modification. Therefore, this study aims to identify m6A modified lncRNAs and their functional role in pancreatic cancer. We performed methylated RNA immunoprecipitation (MeRIP) profiling against a focused group of lncRNAs and identified five lncRNA candidates, among which the highly enriched lncRNA was LINC00901. RNA dot blot, m6A RNA methylation quantification detection and in vivo RNA pulldown combined with m6A RNA methylation Western Blot confirmed the m6A modification of LINC00901. Of note, the analysis of TCGA datasets indicated that the high level of LINC00901 is associated with the poor overall survival (OS) of pancreatic ductal adenocarcinoma (PDAC) patients. Consistent with this result, we showed that LINC00901 promotes pancreatic cancer cell growth. Importantly, we identified that YTHDF1 serves as a “reader” of the m6A modified LINC00901 and furthermore, ectopic expression of YTHDF1 causes downregulation of LINC00901, suggesting a role for m6A modification in regulation of LINC00901 expression. Experiments are underway to determine how LINC00901 promotes pancreatic cancer cell growth. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. N6-Methyladenosine modification of LINC00901 promotes pancreatic cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4827.

Pancreatic ductal adenocarcinoma (PDAC), a most challenging malignant disease, is estimated to become the second leading cause of cancer-related deaths in the United States between the year 2020 and 2030. Although the aberrant activation of KRAS has been demonstrated as the driving force of PDAC progression, the contribution of other molecules, especially long non-coding RNAs (lncRNAs), to PDAC progression remain largely unknown. In the present study, we interrogated the pancreatic adenocarcinoma dataset of the Cancer Genome Atlas (TCGA) consisting of 186 cases. Among 2,730 lncRNAs analyzed, LINC00346 revealed the highest alterations among these lncRNAs with 11% frequency at the RNA level. In addition, its upregulation was significantly associated with overall survival (OS) and disease-free survival (DFS), respectively. To determine the biological function of LINC00346 in pancreatic cancer, we next performed loss-of-function assay in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 by CRISPR/Cpf1 technology using dual gRNA approach. The results showed that knockout (KO) of LINC00346 impaired pancreatic cancer cell proliferation, tumorigenesis, migration and invasion ability, while these phenotypes were restored by LINC00346 re-expression in KO cells (i.e., rescue experiment). Mechanistically, LINC00346 functions as a positive regulator of c-Myc. Further characterizations demonstrated that LINC00346 transcriptionally activates c-Myc expression by interaction with CTCF. Together, these results suggest that LINC00346 contributes to PDAC pathogenesis by activating c-Myc, and thus, LINC00346 may serve as a potential target for pancreatic cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Wan-Xin Peng, Yin-Yuan Mo. LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1821.

Triple-negative breast cancer (TNBC) is responsible for a majority of the mortalities through aggressive metastatic spread, which accounts for a poor 5-year survival. Despite progress in the development of drugs that efficiently target cancer cells, treatments for TNBC are often ineffective. Increasing evidence from epidemiologic studies indicate that cigarette smoking is a major risk factor of TNBC with increased metastatic burden and recurrence. Nicotine, the major addictive component of cigarettes, is not carcinogenic but can promotes cancer progression. However, how nicotine promotes breast cancer lung metastasis is not well understood. To address this question, we orthotopically implanted 4T1 cells (TNBC) into Balb/c mice followed by i.p. injection of nicotine and measured tumor growth and metastatic progression. We found that nicotine increased the growth of primary tumor by 10-fold while it promoted the lung metastasis burden by 100-fold in vivo. Similar results were obtained in experimental metastasis. Immunohistochemical analysis of the primary and lung metastasis tissues showed predominant infiltration of neutrophils in lung metastasis lesions. This observation was further validated by depleting neutrophils using anti-Ly6G antibody, which showed significant decrease in lung metastasis burden in vivo. Intriguingly, infiltrated neutrophils in the lung metastasis tissues selectively overexpressed N2-polarization phenotype. Furthermore, in vitro treatment of primary neutrophils with nicotine induced them into N2 phenotype in a Stat3-dependent manner. To explore further the functional contribution of neutrophils to metastasis, we cultured MDA-MB-231 and MCF10CA (TNBC) cells in primary neutrophils conditioned medium (CM) prior treated with or without nicotine. We found that both MDA-MB-231 and MCF10CA cells displayed epithelial phenotype with loss of mesenchymal markers and gain of epithelial markers in nicotine-induced primary neutrophils CM. These results strongly suggest that nicotine-induced polarization of N2-neutrophils plays a critical role in regulating cancer cell plasticity. We then examined secretory factor(s) from nicotine-treated primary neutrophils using a customized qPCR array system. We found that secretion of LCN2 was significantly augmented in nicotine treated primary neutrophils conditioned medium. In addition, clinical data of stage-II breast cancer patients also showed high LCN2 serum level in smokers than non-smokers. Collectively, our results suggest that nicotine promotes N2 polarization of neutrophils thereby supporting MET transition in a paracrine manner via secreting LCN2 that facilitate metastatic colonization of TNBC cells. Citation Format: Abhishek Tyagi, Sambad Sharma, Kerui Wu, Shih-Ying Wu, Fei Xing, Yin Liu, Dan Zhao, Ravindra Pramod Deshpande, Yin-Yuan MO, Kounosuke Watabe. Nicotine promotes lung metastasis in triple negative breast cancer through paracrine signaling in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2025.

Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24−/CD44+/ESA+) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer. Cancer Res; 73(4); 1434–44. ©2012 AACR.

5-aza-2'-deoxycytidine (decitabine; DAC) is an inhibitor of DNA methyltransferases which catalyze the transfer of a methyl group to DNA. It is well known that aberrant hypermethylation occurs in many types of tumors and often contributes to suppression of important genes such as tumor suppressor genes. Reactivation of the methylated genes by DAC has been shown to induce cytotoxicity and cell cycle dynamics. Increasing evidence indicates that lncRNAs may play critical roles in cancer development through epigenetic regulation of gene expression. However, little is known whether and how lncRNAs are involved in regulation of methylation pathways. In present study, we applies the CRISPR/Cas9-based synergistic activation mediator (SAM) system to a DAC-induced cytotoxicity model to identify potential lncRNAs capable of conferring the resistance to DAC in breast cancer. This screen identified several potential lncRNA candidates and among them is NCRMS which is capable of promoting resistance to DAC. We further confirmed that ectopic expression of NCRMS causes resistance to DAC whereas knockout of NCRMS increases the sensitivity to DAC. Mechanistically, NCRMS functions as a positive regulator of DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B). Further characterizations demonstrated that NCRMS promotes the stability of DNMT3B mRNA. Together, these results suggest that NCRMS may serve as a potential target for cancer therapy. Studies are underway to delineate the detailed mechanism of how NCRMS regulates the stability of DNMT3B mRNA. Citation Format: Wan-xin Peng, Yin-yuan Mo. The regulation of DNMT3B expression by lncRNA-NCRMS in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1397.

The fatty acid synthase (FAS) gene is significantly up-regulated in various types of cancers, and blocking the FAS expression results in apoptosis of tumor cells. Therefore, FAS is considered to be an attractive target for anticancer therapy. However, the molecular mechanism by which the FAS gene is up-regulated in tumor cells is poorly understood. We found that FAS was significantly up-regulated by hypoxia, which was also accompanied by reactive oxygen species (ROS) generation in human breast cancer cell lines. The FAS expression was also activated by H2O2, whereas N-acetyl-l-cystein, a ROS inhibitor, suppressed the expression. We also found that the hypoxia significantly up-regulated sterol regulatory–element binding protein (SREBP)-1, the major transcriptional regulator of the FAS gene, via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF1). Moreover, our results of reporter assay and chromatin immunoprecipitation analysis indicate that SREBP-1 strongly bound to the SREBP binding site/E-box sequence on the FAS promoter under hypoxia. In our xenograft mouse model, FAS was strongly expressed in the hypoxic regions of the tumor. In addition, our results of immunohistochemical analysis for human breast tumor specimens indicate that the expressions of both FAS and SREBP-1 were colocalized with hypoxic regions in the tumors. Furthermore, we found that hypoxia-induced chemoresistance to cyclophosphamide was partially blocked by a combination of FAS inhibitor and cyclophosphamide. Taken together, our results indicate that FAS gene is up-regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP-1 gene, and that hypoxia-induced chemoresistance is partly due to the up-regulation of FAS. [Cancer Res 2008;68(4):1003–11]

Prostate cancer is the second leading cause of death in men in the USA. Despite the high initial response rates to androgen deprivation therapy (ADT), these tumors ultimately develop the resistance, i.e., castration-resistant prostate cancer (CRPC). One of the contributing factors is constitutive expression of androgen receptor (AR) splice variants such as AR3, a major splice variant identified in clinical specimens. However, little is known about long non-coding RNAs (lncRNAs) on regulation of AR splicing. In this study, we generate CRISPR/Cas9-based libraries against a focus group of lncRNAs and an AR3 mini-gene reporter to identify lncRNAs involved in regulation of AR splicing. The principles of this screening system is that only sgRNAs capable of promoting AR3 expression will be enriched in SAM (Synergistic Activation Mediator) library, whereas these clones are lost from the knockout gRNA library after selection. This screen identifies several candidate lncRNA sgRNAs and dual gRNAs (targeting Lnc-DC/ Har1B/ LINC00568/ DHRS4-AS1/ SNHG5). Further characterization confirms the regulation of their corresponding endogenous genes and AR3 expression. Importantly, overexpression of Lnc-DC increases AR3 level and enhances tumor cell growth in response to androgen deprivation. Moreover, while ectopic expression of DHRS4-AS1 increases, DHRS4-AS1 knockout suppresses AR3. Finally, rescue experiments restore the ability of DHRS4-AS1 to induce AR3, further supporting a role for Lnc-DC and DHRS4-AS1 in AR3 regulation. Together, we demonstrate that the CRISPR/Cas9-based screening system provides a powerful tool for lncRNA research. As a result, these lncRNAs may serve as biomarkers to predict the response to ADT or therapeutic targets for treatment of CRPC. Citation Format: Tsui-Ting Ho, Pratirodh Koirala, Nanjiang Zhou, Yin-Yuan Mo. Identification of lncRNAs involved in regulation of androgen receptor splice variants in prostate cancer through CRISPR-Cas9-based screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2017-3046

The human genome expresses a large number of long non-coding RNAs (lncRNAs) and increasing evidences suggest their physiological functions.However, the roles vast majority of lncRNAs are elusive in breast cancer. In the present study, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues as compared to normal tissue. AK023948 is one of such lncRNAs which is up-regulated in breast cancer tissue and in breast cancer cell lines. Ectopic expression of AK023948 in breast cancer MCF-7 cells promotes proliferation and anchorage independent cell growth along with activation of AKT phosphorylation. On the other hand, knockdown or knockout of AK023948 in MCF-7 cells reduces the proliferation of cells and phosphorylation of AKT. Furthermore, there is a positive correlation between AK023948 and pAKT expression in breast cancer tissue microarray. To determine the underlying mechanism of AK023948-mediated activation of AKT, we performed RNA precipitation assays using biotin-labeled AK023948 RNA probe combined with mass spectrometry analysis and identified that ATP dependent RNA helicase A (RHA/DHX9) is AK023948. The interaction of AK023948 with DHX9 was confirmed by Western blot and RNA immunoprecipitation (IP), respectively. Co-IP indicated that DHX9 interacts with PI3K regulatory subunit p85. Further characterization revealed that AK023948 works as scaffold for p85-DHX9 interaction, which is essential for stability of p85 and AKT activity. Finally, we showed that AK023948 is essential to AKT activation induced by growth factors and acidosis, but it has no effect on activation of ERK phosphorylation, suggesting that AK023948 specifically regulate AKT signaling via PI3K. Together, these results suggest that AK023948 contributes to breast cancer pathogenesis by activating AKT and AK023948 may serve as a novel target for breast cancer therapy. Citation Format: Pratirodh Koirala, Yin Yuan Mo. A novel function of LncRNA AK023948—regulation of PI3K signaling. [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 989.

PCGEM1 has been shown to be prostate cancer specific long non-coding RNA (lncRNA). Our previous study indicates that PCGEM1 plays a role in castration resistance through regulation of androgen receptor (AR) splice variants. Although it is known that PCGEM1 is often upregulated in prostate cancer, little is known how PCGEM1 is regulated. In the present study, we show that p54/nrb is a positive regulator for PCGEM1. PCGEM1 is higher in castration resistant CWR22Rv1 than in androgen sensitive LNCaP cells. Moreover, androgen deprivation induces PCGEM1 in LNCaP cells. Histone ChIP assays suggest that the active binding site is within 500 bp upstream of putative transcriptional start site. By using biotin-labelled PCR product and pulldown assays combined with mass spectrometry analysis, we show that p54/nrb interacts with the promoter of PCGEM1. While ectopic expression increases, suppression of p54/nrb by RNAi or knockout (KO) suppresses PCGEM1, supporting a role for p54/nrb in PCGEM1 regulation. Moreover, rescue experiments indicate that re-expression of p54/nrb in KO cells restores the ability to induce PCGEM1. Finally, 3, 3’-Diindolylmethane (DIM), a known chemoprevention agent derived from plants such as broccoli, is capable of suppressing PCGEM1 expression by preventing the interaction of p54/nrb with the PCGEM1 promoter. In particular, DIM reduces tumor growth by suppression of PCGEM1 and promoting apoptosis in the castrated xenograft mouse model. Together, these results demonstrate a novel mechanism of how PCGEM1 and AR splicing are regulated. As a result, PCGEM1 may ultimately contribute to castration resistance. Citation Format: Tsui-Ting Ho, Yin-Yuan Mo. Regulation of PCGEM1 in castration resistant prostate cancer. [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 957.

MicroRNAs are important gene regulators that could play a profound role in tumorigenesis. Our previous studies indicate that miR-145 is a tumor suppressor capable of inhibiting tumor cell growth both in vitro and in vivo. In this study, we show that miR-145 exerts its function in a cell-specific manner. Although miR-145 inhibits cell growth in MCF-7 and HCT-116 cells, it has no significant effect on cell growth in metastatic breast cancer cell lines. However, miR-145 significantly suppresses cell invasion in these cells; in contrast, the antisense oligo against miR-145 increases cell invasion. miR-145 is also able to suppress lung metastasis in an experimental metastasis animal model. This miR-145–mediated suppression of cell invasion is in part due to the silencing of the metastasis gene mucin 1 (MUC1). Using luciferase reporters carrying the 3′-untranslated region of MUC1 combined with Western blot and immunofluorescence staining, we identify MUC1 as a direct target of miR-145. Moreover, ectopic expression of MUC1 enhances cell invasion, which can be blocked by miR-145. Of interest, suppression of MUC1 by miR-145 causes a reduction of β-catenin as well as the oncogenic cadherin 11. Finally, suppression of MUC1 by RNAi mimics the miR-145 action in suppression of invasion, which is associated with downregulation of β-catenin and cadherin 11. Taken together, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor growth but also cell invasion and metastasis. Cancer Res; 70(1); 378–87

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system (CRISPR/Cas) has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines. Citation Format: Tsui-Ting Ho, Nanjiang Zhou, Jianguo Huang, Pratirodh Koirala, Min Xu, Roland Fung, Fangting Wu, Yin-Yuan Mo. Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2015-167

Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is found up-regulated in several tumors. However, little is known about the role of MALAT1 in breast cancer, which is one of the highly invasive tumors among women worldwide. In this study, we found that MALAT1 was highly expressed in both breast cancer cell lines and breast cancer specimens in comparison to normal specimens. We found that low extracellular pH (pHe-acidosis), a major hallmark of solid tumors, further up-regulates MALAT-1 in several breast cancer cell lines in a time-dependent manner. Furthermore, acidosis induces epithelial to mesenchymal transition (EMT)-associated vimentin and Twist 1 expression but suppresses E-cadherin levels in breast cancer cells. Moreover, breast cancer cells under low pHe adopt stem-ness property as revealed by an increase in CD44+ and ESA+ population, mammosphere formation, and expression of OCT-4, SOX-2 and Nanog. Acidosis promotes translocation of β-catenin from cytoplasm to nucleus. The antisense oligonucleotides mediated gene silencing of MALAT-1 prevents acidosis induced nuclear translocation of β-catenin. This is in consistent with our previous finding that breast cancer cells cultured under low pHe exhibit an increased invasion activity. These results suggest that acidosis-induced MALAT-1 trigger an EMT program, leading to cell invasion and metastasis. Therefore, MALAT-1 represents a potential target for breast cancer therapy. Studies are underway to delineate the mechanistic association between MALAT-1, β-catenin, and EMT in acidic microenvironment. Citation Format: Subash C. Gupta, Yin-Yuan Mo. MALAT1 is crucial for epithelial-mesenchymal transition of breast cancer cells in acidic microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 168. doi:10.1158/1538-7445.AM2015-168

The human genome expresses a large number of long non-coding RNAs (lncRNAs), however, little is known about their physiological functions or their role in cancer for the vast majority of these lncRNAs. In the present study, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues as compared to normal tissue. Among them is AK023948 which is up-regulated in breast cancer tissue and in breast cancer cell lines. Ectopic expression of AK023948 in breast cancer MCF-7 cells promotes proliferation and anchorage independent cell growth along with activation of AKT phosphorylation. On the other hand, knockdown or knockout of AK023948 in MCF-7 cells reduces the proliferation of cells and phosphorylation of AKT. Furthermore, there is a positive correlation between AK023948 and pAKT expression in breast cancer tissue microarray. To determine the underlying mechanism of AK023948-mediated activation of AKT, we performed RNA precipitation assays using biotin-labeled AK023948 RNA probe, followed by PAGE analysis, and detected a unique protein band by silver staining. Mass spectrometry analysis identified it as ATP dependent RNA helicase A (RHA/DHX9) which was confirmed by Western blot and RNA immunoprecipitation (IP), respectively. Co-IP indicated that DHX9 interacts with p85PI3K regulatory subunit. Further characterization revealed that p85-DHX9 interaction is essential for stability of p85α and AKT phosphorylation. Together, these results suggest that AK023948 may function as an oncogenic lncRNA in breast cancer and thus, it may serve as a novel target for breast cancer therapy. Citation Format: Pratirodh Koirala, Yin Yuan MO. LncRNA AK023948 promotes breast tumorigenesis by enhancing AKT phosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2015-155

LncRNA BC200 is a small functional RNA which has been implicated in the regulation of protein synthesis in neurons. It is dysregulated in invasive carcinomas of the breast. In this study, we reported that BC200 is upregulated in breast cancer, as detected by RT-PCR analysis of breast cancer tissue scan, suggesting its possible role as an oncogene. We then demonstrated that BC200 is transcriptionally induced by estradiol (E2) treatment in ER positive breast cancer cell lines through multiple functional estrogen response elements (EREs), including the full ERE sequence ∼800bp from transcriptional start site. Luciferase reporter assay combined with mutation analysis indicated that the full ERE region is critical to the estradiol-induced promoter activity. Furthermore, BC200 is crucial for cell growth and viability; its knockdown induces apoptosis in MCF-7 cells. This may explain in part why estrogen can promote cell proliferation in ER positive breast cancer cells. To better study its role in breast cancer development and progression, we generated BC200 knockout MCF-7 cell cells using CRISPR/Cas9 technique. BC200 knockout leads to the release of cytochrome c from mitochondria and promotes the cleavage of PARP-1, both of which are an indicator of cell apoptosis. This was further confirmed by TUNEL assay. It has been reported that hnRNP A2/B1 alters the bcl-X pre-mRNA splicing in favor of anti-apoptotic bcl-XL. Of great interest, BC200 knockout enhances expression of pro-apoptotic bcl-XS form at both mRNA and protein level. RNA pulldown assays using biotin-labeled BC200 probe suggested that BC200 can bind to hnRNP Q and hnRNP A2/B1. On the other hand, RNA immunoprecipitation using hnRNP A2/B1 antibody in BC200 knockout MCF-7 cells showed a significant reduction in enrichment of bcl-X mRNA as compared to control cells. We propose that the regulation of bcl-X alternative splicing depends upon endogenous expression of BC200 that would help hnRNP A2/B1 to position itself on bcl-X mRNA and modulate splicing in favor of bcl-XL. Thus, loss of BC200 leads to generation of bcl-XS protein synthesis and to apoptosis. Together, these results suggest that BC200 functions as a negative regulator of apoptosis in breast cancer and thus, it may serve as a potential target for cancer therapies. Citation Format: Ramesh Singh, Yin-yuan Mo. LncRNA BC200 is induced by estradiol and regulates apoptosis in MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2015-156

Ubc9 is an E2-conjugating enzyme required for sumoylation and has been implicated in regulating several critical cellular pathways. We have shown previously that Ubc9 is important for sumoylation and nucleolar delocalization of topoisomerase (topo) I in response to topo I inhibitors such as topotecan. However, the role for Ubc9 in tumor drug responsiveness is not clear. In this study, we found that although MCF7 cells expressing a Ubc9 dominant-negative mutant (Ubc9-DN) display decreased activity of topo I, these cells are more sensitive to the topo I inhibitor topotecan and other anticancer agents such as VM-26 and cisplatin. In addition, we found that alteration of Ubc9 expression correlates with drug responsiveness in tumor cell lines. To understand possible mechanisms of Ubc9-associated drug responsiveness, we examined several proteins that have been shown to interact with Ubc9 and that may be involved in drug responsiveness. One such protein is Daxx, which is a Fas-associated protein that plays a role in Fas-mediated apoptosis by participating in a caspase-independent pathway through activation of apoptosis signal-regulating kinase 1 and c-Jun NH2-terminal kinase. We found that cells expressing Ubc9-DN accumulate more cytoplasmic Daxx than the control cells. Because cytoplasmic Daxx is believed to participate in cellular apoptosis, we suggest that the interaction of Ubc9 with Daxx and subsequent alteration in the subcellular localization of Daxx may contribute to the increased sensitivity to anticancer drugs in the cells expressing Ubc9-DN. Finally, we found that overexpression of Daxx sensitizes cells to anticancer drugs possibly in part through alterations of the ratio of cytoplasmic and nuclear Daxx. Together, our results suggest a role for Ubc9 in tumor drug responsiveness.

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a bacterial adaptive immune defense against foreign nucleic acids from viruses, plasmids, and other mobile genetic elements. There are three types of CRISPR systems; type II uses single effector enzyme, Cas9, to cleave double-stranded DNA (dsDNA). Because of this unique feature, CRISPR/Cas9 system has been widely used as a genetic engineering tool from bacteria to mammals. In this system, Cas9 nuclease can bind and cleave genome directed by sequence-specific guide RNA (gRNA). While the wild type Cas9 can cleave double stranded DNA, the mutant Cas9 at D10A and H804A can only bind to but cannot cleave the DNA. Given this unique feature, we tried to repurpose Cas9 as a gene activation or suppression tool. To achieve this, we generated DMCas9 (double-mutant Cas9), and fused it with VP16AD (VP16 activation domain) or VP64AD (VP64 activation domain) to explore its utility for gene activation. To generate a Cas9-based gene silencing tool, we fused DMCas9 with Krab, a well-known protein domain with suppression function. Using a reporter system carrying a specific sequence that was recognized by a given gRNA and mCherry as a readout, we demonstrated that co-expression of VP16AD-DMCas9 and specific gRNAs leads to a at least three-fold induction of mCherry signal; in contrast, DMhCas9-Krab along with the specific gRNA resulted in a significant suppression of mCherry activity. Furthermore, we showed that DMCas9-Krab combined with gRNAs suppressed endogenous expression of AR and ER in MCF-7 cells, causing cell growth inhibition. Together, these results demonstrate the feasibility of Cas9-based gene activation and gene suppression. Citation Format: Nanjiang Zhou, Yin-Yuan Mo. Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2014-461

Background: Lysophospholipid receptors (LPRs), including Lysophosphatidic acid (LPA) receptors and sphingosine 1-phosphate (S1P) receptors, are a group of G protein-coupled receptors. LPRs are involved in a wide range of biological processes, such as embryogenesis, systemic development, immune cell trafficking, and inflammatory reactions. The emerging evidence has suggested that the activities of LPRs may be associated with pathogenesis and metastasis of several human cancers. In this study, the expression levels of several LPRs were determined by immunohistochemistry (IHC) in various benign and malignant tissues from various human system/organs, in order to correlate the expression levels of these LPRs with different malignancies. Methods: IHC for EDG1 (S1P receptor 1, S1P1, C- and N-terminals), EDG8 (S1P receptor 5, S1P5), EDG2 (LPA receptor 1, LPA1) and EDG4 (LPA receptor 2, LPA2) was performed on tissue microarray slides containing 384 formalin-fixed paraffin-embedded benign and malignant tissues from 33 human system/organs. The expression level of each LPR was reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3) with a maximal score of 9. Student t-test was employed to analyze the differences in IHC score of each studied LPR between benign and malignant tissues in overall and in individual system/organ. Results: The IHC signals for all studied LPRs were seen in both cytoplasm and nuclei of most tissues. In the level of overall benign and malignant tissues, the expression pattern was similar for all studied LPRs: the cytoplasmic IHC signals were decreased, but the nuclear IHC signals were increased in malignant tissues as compared with benign tissues. Among them, the expression level of cytoplasmic S1P1 (C-terminal) was significantly decreased, and expression levels of nuclear S1P1 (N-terminal) and LPA1 were significantly increased in overall malignant tissues as compared with overall benign tissues. At the organ level, a significant increase in nuclear expression was observed in liver malignancies for all studied LPRs, whereas a decrease in nuclear expression was observed in lung malignancies for all studied LPRs. In colon malignancies, the expression levels of S1P1 (both N- and C-terminals) were significantly increased, but the expression level of LPA1 was significantly increased as compared with benign colon tissues. Conclusion: The constitutive expression of cytoplasmic LPRs may be essential for physiological processes in all tissues. However alterations in nuclear expression of studied LPRs may differentially correlate with the tumorogenesis in different system/organs. Citation Format: Chunyi Wang, Samantha Redfield, Jinghe Mao, Yinyuan Mo, Xinchun Zhou. Differential expressions of lysophospholipid receptors (LPRs) in benign and malignant tissues from various human system/organs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3011. doi:10.1158/1538-7445.AM2014-3011

Long non-coding RNA (lncRNA)-21A is a polymerase III driven non-coding RNA consisting of 324 nucleotides in length. LncRNA-21A was originally identified through in silico analysis and its expression was then confirmed in human cell lines. Further study suggested that lncRNA-21A can control the proliferation of human tumor cell lines, possibly through regulation of CENP-F expression. However, little is known whether lncRNA-21A plays a role in breast cancer. In this study, we reported that lncRNA-21A is downregulated in breast cancer, as detected by RT-PCR analysis of breast cancer tissue scan, suggesting its possible role as a tumor suppressor. In consistent with this finding, lncRNA-21A promotes apoptosis and attenuates cell invasion ability in breast cancer cells. Western blot revealed that overexpression of lncRNA-21A can reduce the phosphorylation of Akt in MCF-7 cells. To determine its underlying mechanism, we performed RNA precipitation assays with the synthetic biotin labeled lncRNA-21A RNA probe followed by mass spectrometry analysis, and we identified hnRNP U and EWS as binding partners for lncRNA-21A. Suppression of EWS by RNAi leads to a reduced level of phosphorylated Akt, suggesting its role in lncRNA-21A-medited Akt activity. Of great interest, we found that like microRNAs, lncRNA-21A can also be secreted through exosomes into conditioned medium. Furthermore, the secreted lncRNA-21A from normal breast epithelial cells can be functionally taken up by recipient metastatic breast cancer cells, suppressing the cell invasion. Together, these results suggest that as a potential tumor suppressor, lncRNA-21A may be used by the host through exosome-mediated gene transfer as a defense mechanism against invasive tumor cells. Citation Format: Ramesh Singh, Yin-yuan Mo. LncRNA-21a is a potential tumor suppressor in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3555. doi:10.1158/1538-7445.AM2014-3555

Background: The human genome makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs. Compared to well-characterized microRNAs, much less is known about lncRNAs. However, emerging evidence has implicated lncRNAs as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. In particular, several studies suggest that lncRNAs can modulate the concentration and biological functions of microRNAs through a competing endogenous RNA (ceRNA) mechanism. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage and miR-145 is a direct transcription target for p53. Therefore, it is important to further characterize this p53 regulatory network involving lncRNA-RoR and miR-145, and to determine their role in tumorigenesis. Methods: To determine cell growth, we performed MTT assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between lncRNA-RoR and the key component (AGO2) of the RNA-induced silencing complex (RISC) was confirmed by RNA co-immunoprecipitation and RNA precipitation. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and miR-145, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model. Results: LncRNA-RoR suppressed miR-145 and upregulated c-Myc, one of major miR-145 target genes. In contrast, RoR-siRNA increased miR-145 expression and inhibited c-Myc. Of interest, miR-145 can also repress lncRNA-RoR expression. This reciprocal repression between lncRNA-RoR and miR-145 appeared to work through the CeRNA mechanism. Mechanistically, we showed that lncRNA-RoR directly interacted with AGO2, a key component of RISC complex, by RNA precipitation assays. This interaction was further confirmed by RNA immunoprecipitation with AGO2 antibody. In consistent with these finding, lncRNA-RoR stimulated proliferation in different cancer cells. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, experiments with dual FISH revealed an inverse correlation between lncRNA-RoR and miR-145 in colon cancer specimens. LncRNA-RoR was upregulated in colon cancer whereas miR-145 was downregulated in the same specimens. Conclusion: Taken together, these results suggest that lncRNA-RoR plays an oncogene role in colon cancer in part through suppression of the tumor suppressor miR-145. Thus, a better understanding of the p53-RoR-miR-145 regulatory system will provide new insight of colon cancer therapy. Citation Format: Jianguo Huang, Yin-Yuan Mo. Suppression of miR-145 by long noncoding RNA RoR in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2014-3537

Although acidic microenvironment has been previously shown to promote breast cancer growth and invasion, the underlying mechanism remains largely unknown. Because inflammation is a major culprit in cancer progression, we hypothesize that pro-inflammatory transcription factor nuclear factor (NF)-κB in the acidic microenvironment plays a major role in the invasion and metastasis of breast cancer. In this study we showed that acidosis (pH 6.6) activates NF-κB but it has no effect on STAT3 activity in breast cancer cells. Mechanistically, the activation of NF-κB is mediated through phosphorylation and degradation of IκBα; nuclear translocation, and phosphorylation of p65, leading to enhanced cell invasion. Upstream to NF-κB signaling, AKT, PDK1, and ERK1/2 are also activated under acidic condition. Moreover, acidosis induces reactive oxygen species (ROS) which can be suppressed by ROS scavengers such as glutathione and N-acetyl-L-cysteine, reversing the acidosis-induced activation of AKT, ERK1/2 and NF-κB, and invasiveness. In addition, the NADPH oxidase (NOX) inhibitor diphenyleneiodonium can suppressed acidosis-induced ROS. Importantly, we observed all of these effects only in cancer cells, but not in non-malignant cells. Together, these results suggest that production of ROS, activation of AKT, ERK1/2 and NF-κB may constitute key acidosis-induced signaling cascades in breast cancer cells. Citation Format: Subash C. Gupta, Yin-Yuan Mo. Acidosis-induced NF-κB promotes cell invasion in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1101. doi:10.1158/1538-7445.AM2014-1101

Introduction: Exosomes are 30-100nm membrane vesicles of endocytic origin containing proteins, mRNAs, microRNAs and lincRNAs. MicroRNAs are small and lincRNAs are large intergenic non-coding RNAs. They are emerging as key regulators of diverse cellular processes including cancer. In this study, we investigated the exosome-mediated transfer of microRNAs and lincRNAs, regulation and functional significance in breast cancer. Approach: Exosomes were isolated by ultracentrifugation and stained with PKH26 dye for cellular uptake in different cells by confocal microscopy. This uptake was also shown by exosome isolated from stable 293T cell line overexpressing the pCDH-CD63-GFP fusion protein. To determine the effect of nSMase2 on exosome secretion, stable cell lines were established with smpd3 gene. Levels of microRNAs and lincRNAs secreted in media were determined by real time PCR and their function validated through western blot and cell invasion. Results: We first showed that exosomes can be transferred among different cell lines through direct uptake. We found that miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cells. Also, nSMase2 promoted the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppressed this secretion. Importantly, western blot revealed that upon uptake, miR-10b can still reduce the protein level of its target genes. We also found lincRNA 21A and BC200 to be secreted through exosome in cell type dependent manner with functional significance. Conclusion: We found that the exosomes secreted from cancer cells containing microRNA and lincRNA can be taken up by other cells and have functional role in the recipient cells suggesting the importance of exosome-mediated RNA transfer in tumor microenvironment. Citation Format: Ramesh Singh, Yin-Yuan Mo. Exosomal non-coding RNAs as mediators for cell-cell communications . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2013-1837

Protein-coding genes account for only a small part of the human genome whereas the vast majority of transcripts make up the non-coding RNAs including microRNAs and long non-coding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, microRNAs can target a large number of protein-coding genes. However, it remains to be determined whether microRNAs can also target lncRNAs. In the present study, we determined whether miR-21 impacts lncRNA expression. Using lncRNA RT-PCR array carrying 83 human disease related lncRNAs, we showed that miR-21 was capable of repressing the growth arrest specific 5 (GAS5) lncRNA which was previously shown to be involved in growth arrest. This negative regulation between miR-21 and GAS5 was confirmed in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. While ectopic expression of GAS5 suppressed, GAS5 siRNAs increased miR-21 expression. Importantly, there was a putative miR-21 binding site in exon 5 of GAS5; deletion of the miR-21 binding site abolished this activity. We further showed that biotin labeled GAS5 RNA probe was able to pull down the RNA-induced silencing complex (RISC) and subsequently identified miR-21 in this GAS5-RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results support the “endogenous competitive RNA” concept that microRNAs may regulate not only protein-coding genes, but also non-coding genes such as GAS5. Citation Format: Ziqiang Zhang, Yin-yuan Mo. Negative regulation of lncRNA GAS5 by miR-21. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2013-1829

Background: The human genome also makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs in addition to protein-coding genes. While microRNAs are well characterized, little is known about lncRNAs. Emerging evidence has implicated lncRNAs in gene regulation; and they are often aberrantly expressed in a variety of human diseases, in particular cancer. The heterogeneous nuclear ribonucleoprotein I (hnRNP I) is a member of the large hnRNP family and has been implicated in mRNA splicing, stability and translational regulation of certain RNA transcripts. During screening for lncRNA-interacted proteins, we identified hnRNP I as a binding partner for UCA1. However, it remains unclear how hnRNP I interacts with UCA1 and whether such an interaction impacts UCA1-mediated cell growth and responsiveness to anti-cancer drugs. Methods: To determine cell growth and response to doxorubicin, we performed MTT assays as well as cell counting assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between hnRNP I and UCA1 was confirmed by RNA co-immunoprecipitation and RNA pulldown. RNA stability was determined by treating MCF-7 cells with actinomycin D, followed by real-time RT-PCR. hnRNP I was knock down by transient transfection of hnRNP I siRNAs. Results: We found that UCA1 induced proliferation and increased resistance to doxorubicin in MCF-7 cells. RNA pulldown assays revealed that UCA1 RNA directly interacted with hnRNP I. This interaction was further confirmed by RNA immunoprecipitation by hnRNP I antibody. Real-time RT-PCR revealed that knockdown of hnRNP I by RNAi reduced the level of UCA1 in MCF-7 cells and it also repressed the induction of UCA1 by doxorubicin. Further experiments suggested that hnRNP I impacts the UCA1 stability. We showed that half-life of UCA1 was about 12 h but was decreased to about 6 h in the presence of hnRNP I siRNAs. We further showed that the phosphorylated form of hnRNA I, predominantly in the cytoplasm, was a major form that interacts with UCA1. Conclusion: Taken together, these results suggest that hnRNP I plays a role in regulation of the stability of UCA1. Since previous work implicates the involvement of UCA1 in tumor cell growth and chemoresistance, a better understanding of the underlying mechanism will provide new insight of targeting UCA1 in cancer therapy. Citation Format: Jianguo Huang, Yin-Yuan Mo. HnRNP I interacts with lncRNA UCA1 and regulates its stability. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1827. doi:10.1158/1538-7445.AM2013-1827

Overwhelming evidence has indicated that microRNAs can play a fundamental role in regulation of diverse cellular functions and dysregulation of microRNA expression could lead to a variety of disorders including human cancer. Although significant progress has been made in the past years in discovery of microRNAs and their biogenesis, and their role in many cellular phenotypes, it is not fully understood how microRNAs exert their cellular functions because a single microRNA can have hundreds of targets. Hence, identification of microRNA targets is a critical step toward understanding of molecular mechanisms of microRNA-mediated gene expression in normal and disease processes. Here we reported a selection system for microRNA target identification and validation. We used Sleeping Beauty (SB) transposon system to generate selection vectors. The first generation of SB-based vector is named pSSMT-5/6 which carries tetO-EF1a promoter driving GFP/or mCherry-T2A-Pu and tetR-Krab under CMV promoter. We examined the feasibility of these new constructs in 293T and HeLa cells. Using GFP or m-Cherry along with Pu (puromycin resistance) as a reporter, we found that the construct carrying either a given UTR or microRNA sponge provides a significant selection advantage over the vector alone. For example, we cloned 6 different UTRs into the construct and identified 3 of them, CDH1, p53 and p27, revealed stronger mCherry signals and are more resistant to puromycin. Thus, we will test our microRNA library to identify microRNAs capable of targeting these genes (i.e., one known UTR construct against the microRNA library). Together, these results suggest that this system provides proof of principle. In the future, we will generate a UTR library so that we will be able to test for an individual microRNA against the UTR library to identify its biologically relevant targets. Citation Format: Nanjiang Zhou, Yin-Yuan Mo. A genetic selection system for microRNA targets identification and validation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5286. doi:10.1158/1538-7445.AM2013-5286

The transcription activator-like effectors (TALEs) are a family of virulence factors produced by plant bacterial pathogens, and they are able to regulate expression of specific host genes by binding to the corresponding promoter regions in a sequence-specific manner. The transcription activator-like effector nuclease (TALEN) is made by fusing a nuclease to the DNA binding modules of TALE, which would allow for generation of DNA deletions, knockouts or knockins. Since their discovery, genes from different organisms have been successfully targeted using designer TALEs through assembling sequence specific modules, which is a critical step to make TALE/TALEN vectors. However, current methods of assembling DNA binding modules, such as the golden-gate ligation, are still time consuming and labor intensive. Moreover, they are also technically challenging. In the present study, we adopted a single step approach to assemble TALE modules. For proof of principle, we used p53 binding site (Tgacttgcatgtacatgcc) derived from the lncRNA-RoR promoter for proof of principle. First, we designed four gBlock DNA oligos which carried 30 bp overlapping between two adjacent oligos. Then, we linked them together by PCR and subsequently directly cloned into an expression vector which carries two flanking TALE sequences. To determine the function of this construct, we generated a reporter vector carrying this p53 binding site and mCherry and showed that this newly made construct was able to activate the mCherry reporter. Experiments are underway to test if the p53 specific TALE is able to activate the endogenous lncRNA-RoR. Citation Format: Juan Zhang, Yin-Yuan Mo. A quick and easy way of assembling TALE/TALEN modules for genetic modifications in mammalian cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2013-4224

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the rate of one year survival among these patients remains less than 20%. Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective due to the blood brain barrier (BBB). Despite this clinical importance, the molecular basis of breast tumor metastasis to the brain is poorly understood. Recent active research in microRNA identified a series of this type of molecules that are involved in tumor progression in various tumors as oncogenes and tumor suppressors. In this study, we have isolated RNA from Formalin-Fixed Paraffin-Embedded (FFPE) samples obtained from primary breast tumors and also from brain metastatic lesions followed by microRNA profiling. The results of our profile analysis for these samples revealed that primary tumor samples expressed significantly high level of miR509 compared to brain metastatic lesions. We also found that this microRNA was capable of modulating two essential genes for invasion, RhoC and TNF alpha. Interestingly, high expression of TNF alpha was significantly and inversely correlated to brain metastasis-free survival of breast cancer patients. Importantly, we found that the expression of miR509 significantly suppressed the ability of cancer cells to metastasize to the brain in our animal model. We also examined the expression of TNF alpha in brain-metastatic lesions and found that it was indeed significantly up-regulated. Furthermore, the results of our in vitro experiments indicate that miR509 attenuates the abilities of invasion of breast cancer cells by modulating the RhoC expression. These results suggest that miR509 may serve as a biomarker or therapeutic targets for brain metastasis of breast cancer. Citation Format: Fei Xing, Hiroshi Okuda, Aya Kobayashi, Puspa R. Pandey, Sambad Sharma, Yin-Yuan Mo, Yin Liu, Kerui Wu, Kounosuke Watabe. miR509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF alpha. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2013-3084 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.