Hypoxia-inducible factor-1α-dependent induction of miR122 enhances hepatic ischemia tolerance.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation.

ERK Activation Globally Downregulates miRNAs through Phosphorylating Exportin-5.

MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications.

RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice.

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.

MicroRNA-122 regulates polyploidization in the murine liver.

UNLABELLED: A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ∼90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy, we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Second, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, lifelong depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated overexpression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l, and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. CONCLUSION: Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified; our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization, and these studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis, and disease. (Hepatology 2016;64:599-615).

Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: role of E2F1 and transcription factor dimerization partner 2.

UNLABELLED: c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3ß, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. CONCLUSION: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3ß expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

Hepatic loss of miR-122 predisposes mice to hepatobiliary cyst and hepatocellular carcinoma upon diethylnitrosamine exposure.

Loss of miR-122 causes chronic steatohepatitis and spontaneous hepatocellular carcinoma. However, the consequence of miR-122 deficiency on genotoxic stress-induced liver pathogenesis is poorly understood. Here, we investigated the impact of miR-122 depletion on liver pathobiology by treating liver-specific miR-122 knockout (LKO) mice with the hepatocarcinogen diethylnitrosamine (DEN). At 25 weeks post-DEN injection, all LKO mice developed CK-19-positive hepatobiliary cysts, which correlated with DEN-induced transcriptional activation of Cdc25a mediated through E2f1. Additionally, LKO livers were more fibrotic and vascular, and developed larger microscopic tumors, possibly due to elevation of the Axl oncogene, a receptor tyrosine kinase as a novel target of miR-122, and several protumorigenic miR-122 targets. At 35 weeks following DEN exposure, LKO mice exhibited a higher incidence of macroscopic liver tumors (71%) and cysts (86%) compared to a 21.4% and 0% incidence of tumors and cysts, respectively, in control mice. The tumors in LKO mice were bigger (ninefold, P = 0.015) and predominantly hepatocellular carcinoma, whereas control mice mostly developed hepatocellular adenoma. DEN treatment also reduced survival of LKO mice compared to control mice (P = 0.03). Interestingly, induction of oxidative stress and proinflammatory cytokines in LKO liver shortly after DEN exposure indicates predisposition of a pro-tumorigenic microenvironment. Collectively, miR-122 depletion facilitates cystogenesis and hepatocarcinogenesis in mice on DEN challenge by up-regulating several genes involved in proliferation, growth factor signaling, neovascularization, and metastasis.

Reduced susceptibility of DNA methyltransferase 1 hypomorphic (Dnmt1N/+) mice to hepatic steatosis upon feeding liquid alcohol diet.

BACKGROUND: Methylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Dnmt1 wild type (Dnmt1(+/+)) and hypomorphic (Dnmt1(N/+)) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in Dnmt1(+/+) mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, Dnmt1(N/+) mice were less susceptible to alcoholic steatosis compared to Dnmt1(+/+) mice. Expression of several key genes involved in alcohol (Aldh3b1), lipid (Ppara, Lepr, Vldlr, Agpat9) and xenobiotic (Cyp39a1) metabolism, and oxidative stress (Mt-1, Fmo3) were significantly (P<0.05) altered in Dnmt1(N/+) mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of Agpat9, Lepr, Mt1 and Ppara were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice. CONCLUSIONS/SIGNIFICANCE: Although feeding alcohol diet reduced Dnmtase activity, the loss of one copy of Dnmt1 protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.

Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver.

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia.

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19(+) spleen B cells from Eµ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.

Lyn kinase and ZAP70 are substrates of PTPROt in B-cells: Lyn inactivation by PTPROt sensitizes leukemia cells to VEGF-R inhibitor pazopanib.

We have recently shown that the gene encoding the truncated form of protein tyrosine phosphatase receptor-type O (PTPROt) expressed predominantly in hematopoietic cells is epigenetically silenced in human primary chronic lymphocytic leukemia (B-CLL). To determine whether increased phosphorylation of the PTPROt substrates following PTPROt suppression alters signal transduction pathway(s) that impart a growth advantage to the leukemic lymphocytes, it is critical to discern the key substrates of PTPROt. Here, we used substrate-trapping assay to identify two novel substrates of PTPROt, the tyrosine kinases Lyn and ZAP70. Both Lyn and ZAP70 were dephosphorylated by wild-type PTPROt, but not by its catalytic site (CS) mutant. A critical phosphorylation site in Lyn, Y397, essential for its activity was dephosphorylated by PTPROt. Consequently, the activity of Lyn kinase was compromised when co-expressed with PTPROt-WT compared to vector control or upon co-expression with PTPROt-CS. Ectopic expression of PTPROt in Raji cells reduced phosphorylation of Lyn in the absence of any change in its protein levels. These results have revealed the physiological importance of PTPROt in regulating B-cell receptor signaling at Lyn kinase. Further, ectopic expression of PTPROt also sensitized the cells to the VEGF-R inhibitor Pazopanib.

HOXB13, a target of DNMT3B, is methylated at an upstream CpG island, and functions as a tumor suppressor in primary colorectal tumors.

BACKGROUND: A hallmark of cancer cells is hypermethylation of CpG islands (CGIs), which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI in their promoters, were methylated in multiple colon cancer cell lines but not in normal colon epithelial cells. Further, these genes were reactivated in RKO cells after treatment with 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. COBRA showed that the CGIs encompassing the promoter and/or coding region of DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located approximately 4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated in DNMT1(-/-) HCT cells but was almost methylation free in cells lacking both DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 abolished tumor growth in nude mice. CONCLUSIONS/SIGNIFICANCE: This is the first report that identifies several important tumor suppressors and transcription factors as direct DNMT3B targets in colon cancer and as potential biomarkers for this cancer. Further, this study shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer.

MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib.

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.

Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice.

UNLABELLED: MicroRNAs (miRs) are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of messenger RNAs (mRNAs) at the posttranscriptional level. Aberrant expression of certain microRNAs plays a causal role in tumorigenesis. Here, we report identification of hepatic microRNAs that are dysregulated at early stages of feeding C57BL/6 mice choline-deficient and amino acid-defined (CDAA) diet that is known to promote nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 84 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P < or = 0.01) altered in mice fed CDAA diet for 6, 18, 32, and 65 weeks compared with those fed choline-sufficient and amino acid-defined (CSAA) diet. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated up-regulation of oncogenic miR-155, miR-221/222, and miR-21 and down-regulation of the most abundant liver-specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic phosphatase and tensin homolog (PTEN) and CCAAT/enhancer binding protein beta (C/EBPbeta), respective targets of miR-21 and miR-155, in these mice at early stages. DNA binding activity of nuclear factor kappa B (NF-kappaB) that transactivates miR-155 gene was significantly (P = 0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Furthermore, the expression of miR-155, as measured by in situ hybridization and real-time RT-PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells, whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P = 0.0004) up-regulated in primary human HCCs with a concomitant decrease (P = 0.02) in C/EBPbeta level compared with matching liver tissues. CONCLUSION: Temporal changes in microRNA profile occur at early stages of CDAA diet-induced hepatocarcinogenesis. Reciprocal regulation of specific oncomirs and their tumor suppressor targets implicate their role in NASH-induced hepatocarcinogenesis and suggest their use in the diagnosis, prognosis, and therapy of liver cancer.

PTPROt inactivates the oncogenic fusion protein BCR/ABL and suppresses transformation of K562 cells.

Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). Because the truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells, we tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. Additionally, the catalytically inactive PTPROt acted as a trapping mutant that was also able to inhibit anchorage independence and facilitate apoptosis of K562 cells. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia.

Estrogen-mediated suppression of the gene encoding protein tyrosine phosphatase PTPRO in human breast cancer: mechanism and role in tamoxifen sensitivity.

We have previously demonstrated the tumor suppressor characteristics of protein tyrosine phosphatase receptor-type O (PTPRO) in leukemia and lung cancer, including its suppression by promoter methylation. Here, we show tumor-specific methylation of the PTPRO CpG island in primary human breast cancer. PTPRO expression was significantly reduced in established breast cancer cell lines MCF-7 and MDA-MB-231 due to promoter methylation compared with its expression in normal human mammary epithelial cells (48R and 184). Further, the silenced gene could be demethylated and reactivated in MCF-7 and MDA-MB-231 cells upon treatment with 5-Azacytidine, a DNA hypomethylating agent. Because PTPRO promoter harbors estrogen-responsive elements and 17beta-estradiol (E2) plays a role in breast carcinogenesis, we examined the effect of E2 and its antagonist tamoxifen on PTPRO expression in human mammary epithelial cells and PTPRO-expressing breast cancer cell line Hs578t. Treatment with E2 significantly curtailed PTPRO expression in 48R and Hs578t cells, which was facilitated by ectopic expression of estrogen receptor (ER)beta but not ERalpha. On the contrary, treatment with tamoxifen increased PTPRO expression. Further, knockdown of ERbeta by small interfering RNA abolished these effects of E2 and tamoxifen. Chromatin immunoprecipitation assay showed association of c-Fos and c-Jun with PTPRO promoter in untreated cells, which was augmented by tamoxifen-mediated recruitment of ERbeta to the promoter. Estradiol treatment resulted in dissociation of c-Fos and c-Jun from the promoter. Ectopic expression of PTPRO in the nonexpressing MCF-7 cells sensitized them to growth-suppressive effects of tamoxifen. These data suggest that estrogen-mediated suppression of PTPRO is probably one of the early events in estrogen-induced tumorigenesis and that expression of PTPRO could facilitate endocrine therapy of breast cancer.