Ling-Yueh Hu

A novel estrogen receptor-microRNA 190a-PAR-1-pathway regulates breast cancer progression, a finding initially suggested by genome-wide analysis of loci associated with lymph-node metastasis

To identify microRNAs that are important in regulating breast cancer progression, the present study used data for the 199 961 single-nucleotide polymorphisms (SNPs) in 837 breast cancer patients genotyped in a recent genome-wide association study to identify loci associated with lymph node metastasis (LNM). SNPs tagging the 15q22.2 locus showed a significant association with LNM and miR-190a was found to be the only microRNA in this region. The role of miR-190a in LNM was supported by the findings that increased miR-190a expression inhibited cell migration and invasiveness and that the target of miR-190a was protease-activated-receptor 1 (PAR-1), which is a metastasis promoting protein in several cancers. In addition, the promoter region of miR-190a was defined and found to contain half of an estrogen response element, suggesting that miR-190a is regulated by estrogen receptor (ER) signaling. This was confirmed by the findings that miR-190a expression was activated by 17β-estradiol and that ERα bound directly to this promoter. The importance of this ERα-miR190a-PAR-1 link in breast tumorigenesis is suggested by the findings of (i) an association between genetic polymorphism of the miR-190a-containing region and LNM that is modified by SNPs of PAR-1 and is particularly significant in ERα-positive patients and (ii) a combined effect of ERα and miR-190a expression on tumor grade/cancer stage. More importantly, the level of miR-190a expression in primary breast carcinomas correlated with overall survival. These findings suggest a novel pathway in which ERα signaling regulates miR-190a expression, causing inhibition of PAR-1 expression, correlated with inhibition of cancer metastasis.

Genetic variation in the genome-wide predicted estrogen response element-related sequences is associated with breast cancer development

IntroductionEstrogen forms a complex with the estrogen receptor (ER) that binds to estrogen response elements (EREs) in the promoter region of estrogen-responsive genes, regulates their transcription, and consequently mediates physiological or tumorigenic effects. Thus, sequence variants in EREs have the potential to affect the estrogen-ER-ERE interaction. In this study, we examined the hypothesis that genetic variations of EREs are associated with breast cancer development.MethodsThis case-control study involved 815 patients of Asian descent with incident breast cancer and 821 healthy female controls. A total of 13,737 ERE sites in the whole genome predicted by a genome-wide computational algorithm were blasted with single-nucleotide polymorphism (SNP) sequences. Twenty-one SNPs located within 2,000 bp upstream or within introns 1 and 2 of putative genes and with a minor allele frequency greater than 5% were identified and genotyped. Frequencies of SNPs were compared between cases and controls to identify SNPs associated with cancer susceptibility.ResultsA significant combined effect of rs12539530, an ERE SNP in intron 2 of NRCAM which codes for a cell adhesion molecule, and SNPs of ESR1, the gene coding for ER, on breast cancer risk was found. Interestingly, this combined effect was more significant in women who had experienced a longer period of lifetime estrogen exposure, supporting a hormonal etiology of this SNP in breast tumorigenesis.ConclusionsOur findings provide support for a role of genetic variation in ERE-ESR1 in determining susceptibility of breast cancer development.

Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in ERK-responsive lung cancer

Metastatic spread of cancer cells portends a poor prognosis and mortality for lung cancer patients. Hypoxia-inducible factor-1α (HIF-1α) enhances tumor cell motility by activating the epithelial-to-mesenchymal transition (EMT), which is considered a prerequisite for metastasis. Recent studies of microRNA involvement in cancer invasion and metastasis have highlighted the role of such RNAs in tumor development. However, little work has been done to identify tumor suppressor microRNAs that target HIF-1α to down-modulate the EMT and thereby counteract the aggressiveness and metastasis of lung cancer cells. Here, we identified the 3′-untranslated region of HIF-1α mRNA as a target of miR-622 and established that miR-622-mediated down-modulation of HIF-1α correlates with decreased levels of mesenchymal proteins, including Snail, β-catenin, and vimentin. Functional analyses revealed that increased miR-622 expression inhibited lung cancer cell migration and invasion in vitro. miR-622 also inhibited the genesis of metastatic lung nodules as demonstrated in a lung cancer xenograft model in which nude mice were transplanted with A549 cells expressing miR-622. Mechanistic analyses showed that overexpression of EGF decreased the miR-622 level in A549 cells, and this reduction could be rescued by administrating U0126, an inhibitor of ERK. Moreover, miR-622 overexpression mediated by the transcription factor FOXO3a decreased the invasiveness of lung tumor cells by inhibiting HIF-1α via inactivation of ERK signaling in U0126-treated A549 cells. These findings highlight the pivotal role of the FOXO3a/miR-622 axis in inhibiting HIF-1α to interfere with tumor metastasis, and this information may contribute to development of novel therapeutic strategies for treating aggressive lung cancer.

Initiation of the ATM-Chk2 DNA damage response through the base excision repair pathway

The DNA damage response (DDR) is activated by various genotoxic stresses. Base lesions, which are structurally simple and predominantly fixed by base excision repair (BER), can trigger the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, a DDR component. How these lesions trigger DDR remains unclear. Here we show that, for alkylation damage, methylpurine-DNA glycosylase (MPG) and apurinic/apyrimidinic endonuclease 1, both of which function early in BER, are required for ATM-Chk2-dependent DDR. In addition, other DNA glycosylases, including uracil-DNA glycosylase and 8-oxoguanine glycosylase, which are involved in repairing deaminated bases and oxidative damage, also induced DDR. The early steps of BER therefore play a vital role in modulating the ATM-Chk2 DDR in response to base lesions, facilitating downstream BER processing for repair, in which the formation of a single-strand break was shown to play a critical role. Moreover, MPG knockdown rescued cell lethality, its overexpression led to cell death triggered by DNA damage and, more interestingly, higher MPG expression in breast and ovarian cancers corresponded with a greater probability of relapse-free survival after chemotherapy, underscoring the importance of glycosylase-dependent DDR. This study highlights the crosstalk between BER and DDR that contributes to maintaining genomic integrity and may have clinical applications in cancer therapy.

The Effect of MicroRNA-124 Overexpression on Anti-Tumor Drug Sensitivity

MicroRNAs play critical roles in regulating various physiological processes, including growth and development. Previous studies have shown that microRNA-124 (miR-124) participates not only in regulation of early neurogenesis but also in suppression of tumorigenesis. In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs). We then examined which DNA repair–related genes, particularly the genes of SB repair, were regulated by miR-124. Two SB repair–related genes, encoding ATM interactor (ATMIN) and poly (ADP-ribose) polymerase 1 (PARP1), were strongly affected by miR-124 overexpression, by binding of miR-124 to the 3¢-untranslated region of their mRNAs. As a result, the capacity of cells to repair DNA SBs, such as those resulting from homologous recombination, was significantly reduced upon miR-124 overexpression. A particularly important therapeutic implication of this finding is that overexpression of miR-124 enhanced cell sensitivity to multiple DNA-damaging agents via ATMIN- and PARP1-mediated mechanisms. The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy. These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.

B-Myb Induces APOBEC3B Expression Leading to Somatic Mutation in Multiple Cancers

The key signature of cancer genomes is the accumulation of DNA mutations, the most abundant of which is the cytosine-to-thymine (C-to-T) transition that results from cytosine deamination. Analysis of The Cancer Genome Atlas (TCGA) database has demonstrated that this transition is caused mainly by upregulation of the cytosine deaminase APOBEC3B (A3B), but the mechanism has not been completely characterized. We found that B-Myb (encoded by MYBL2) binds the A3B promoter, causing transactivation, and this is responsible for the C-to-T transitions and DNA hypermutation in breast cancer cells. Analysis of TCGA database yielded similar results, supporting that MYBL2 and A3B are upregulated and putatively promote C-to-T transitions in multiple cancer types. Moreover, blockade of EGF receptor with afatinib attenuated B-Myb–A3B signaling, suggesting a clinically relevant means of suppressing mutagenesis. Our results suggest that B-Myb–A3B contributes to DNA damage and could be targeted by inhibiting EGF receptor.

FGFR2 regulates Mre11 expression and double-strand break repair via the MEK-ERK-POU1F1 pathway in breast tumorigenesis

The association between breast cancer risk and genetic variants of fibroblast growth factor receptor 2 (FGFR2) has been identified and repeatedly confirmed; however, the mechanism underlying FGFR2 in breast tumorigenesis remains obscure. Given that breast tumorigenesis is particularly related to DNA double-strand-break-repair (DSBR), we examined the hypothesis that FGFR2 is involved in DSBR. Our results show that expression of Mre11, a vital exonuclease in DSBR, is downregulated by FGFR2, which is further linked to decreased DSBR. Analysis of the Mre11 promoter revealed that POU1F1 mediates FGFR2-induced Mre11 downregulation. Furthermore, ERK, downstream of FGFR2, directly interacts with and phosphorylates POU1F1, increasing POU1F1 binding capacity to the Mre11 promoter and repressing Mre11 expression, which consequently affects DSBR and sensitizes breast cancer cells to chemotherapeutic treatments. The importance of the FGFR2-Mre11-DSBR link in cancer progression is suggested by the finding that genotypes of FGFR2 and Mre11 are associated with survival of breast cancer patients and that FGFR2 expression correlates with cancer prognosis specifically in patients receiving chemotherapy. This study yields important insight into the role of FGFR2 in breast tumorigenesis and may facilitate development of a useful therapeutic approach for breast cancer.

Functional variants at the 21q22.3 locus involved in breast cancer progression identified by screening of genome-wide estrogen response elements

IntroductionEstrogen forms a complex with the estrogen receptor (ER) that binds to estrogen response elements (EREs) in the regulatory region of estrogen-responsive genes and regulates their transcription. Sequence variants in the regulatory regions have the potential to affect the transcription factor–regulatory sequence interaction, resulting in altered expression of target genes. This study explored the association between single-nucleotide polymorphisms (SNPs) within the ERE-associated sequences and breast cancer progression.MethodsThe ERE-associated sequences throughout the whole genome that have been demonstrated to bind ERα in vivo were blasted against online information from SNP data sets and 54 SNPs located adjacent to estrogen-responsive genes were selected for genotyping in two independent cohorts of breast cancer patients: 779 patients in the initial screening stage and another 888 in the validation stage. Deaths due to breast cancer or recurrence of breast cancer were defined as the respective events of interest, and the hazard ratios of individual SNPs were estimated based on the Cox proportional hazards model. Furthermore, functional assays were performed, and information from publicly available genomic data and bioinformatics platforms were used to provide additional evidence for the associations identified in the association analyses.ResultsThe SNPs at 21q22.3 ERE were significantly associated with overall survival and disease-free survival of patients. Furthermore, these 21q22.3 SNPs (rs2839494 and rs1078272) could affect the binding of this ERE-associated sequence to ERα or Rad21 (an ERα coactivator), respectively, which resulted in a difference in ERα-activated expression of the reporter gene.ConclusionThese findings support the idea that functional variants in the ERα-regulating sequence at 21q22.3 are important in determining breast cancer progression.

Abstract 144: MicroRNA-190, regulated by estrogen receptor signaling, suppresses expression of the metastasis-promoting gene PAR-1 and is associated with breast cancer progression

Cancer metastasis contributes to mortality of breast cancer patients. The present study, based on the fundamental concept that dissociation of the extracellular matrix is the driving force for tumor metastasis, was performed to examine the hypothesis that microRNA-190 (miR-190) is important in regulating breast cancer metastasis by decreasing the expression of protease-activated receptor-1 (PAR-1), a gene encoding a receptor for matrix metalloproteinase1 and thrombin that is associated with tumor metastasis. This hypothesis was initially suggested by the observations that the expression of a reporter gene could be regulated by a specific sequence in the 3′-untranslated region of PAR-1 and that miR-190 was complementary to this sequence. Support for our hypothesis came from the findings that (a) PAR-1 expression was directly inhibited by miR-190, (b) increased miR-190 expression suppressed cell migration and invasiveness, and (c) the level of miR-190 expression in primary breast carcinomas correlated with overall survival. Interestingly, we defined the promoter region of miR-190 and noted that it contained half of an estrogen receptor (ER) response element, supporting the breast tumorigenic contribution of miR-190. This was further confirmed by the findings that miR-190 expression was activated by 17β-estradiol and that the ER bound directly to this promoter and regulated miR-190 expression. The findings of the present study may explain why ER-positive patients usually have a favorable progression and how ER regulates cancer metastasis, i.e. ER signaling regulates miR-190 expression, thus causing suppression of PAR-1 expression, resulting in inhibition of breast cancer metastasis. 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 144. doi:1538-7445.AM2012-144

SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair

XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER.