Mono-(2-ethyl-5-hydroxyhexyl) phthalate promotes uterine leiomyoma cell survival through tryptophan-kynurenine-AHR pathway activation.

Uterine leiomyoma is the most common tumor in women and causes severe morbidity in 15 to 30% of reproductive-age women. Epidemiological studies consistently indicate a correlation between leiomyoma development and exposure to endocrine-disrupting chemical phthalates, especially di-(2-ethylhexyl) phthalate (DEHP); however, the underlying mechanisms are unknown. Here, among the most commonly encountered phthalate metabolites, we found the strongest association between the urine levels of mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), the principal DEHP metabolite, and the risk of uterine leiomyoma diagnosis (n = 712 patients). The treatment of primary leiomyoma and smooth muscle cells (n = 29) with various mixtures of phthalate metabolites, at concentrations equivalent to those detected in urine samples, significantly increased cell viability and decreased apoptosis. MEHHP had the strongest effects on both cell viability and apoptosis. MEHHP increased cellular tryptophan and kynurenine levels strikingly and induced the expression of the tryptophan transporters SLC7A5 and SLC7A8, as well as, tryptophan 2,3-dioxygenase (TDO2), the key enzyme catalyzing the conversion of tryptophan to kynurenine that is the endogenous ligand of aryl hydrocarbon receptor (AHR). MEHHP stimulated nuclear localization of AHR and up-regulated the expression of CYP1A1 and CYP1B1, two prototype targets of AHR. siRNA knockdown or pharmacological inhibition of SLC7A5/SLC7A8, TDO2, or AHR abolished MEHHP-mediated effects on leiomyoma cell survival. These findings indicate that MEHHP promotes leiomyoma cell survival by activating the tryptophan-kynurenine-AHR pathway. This study pinpoints MEHHP exposure as a high-risk factor for leiomyoma growth, uncovers a mechanism by which exposure to environmental phthalate impacts leiomyoma pathogenesis, and may lead to the development of novel druggable targets.

Transcriptional Profiling of Human Endocervical Tissues Reveals Distinct Gene Expression in the Follicular and Luteal Phases of the Menstrual Cycle.

The endocervix plays an important role in providing appropriate protective mechanisms of the upper female reproductive tract (FRT) while at the same time providing the appropriate milieu for sperm transport. Hormone fluctuations throughout the menstrual cycle contribute to changes in the mucosal environment that render the FRT vulnerable to infectious diseases. The objective of this study was to identify genes in human endocervix tissues that were differentially expressed in the follicular versus the luteal phases of the menstrual cycle using gene expression profiling. A microarray using the IIlumina platform was performed with eight endocervix tissues from follicular and four tissues from luteal phases of the menstrual cycle. Data analysis revealed significant differential expression of 110 genes between the two phases, with a P value <0.05 and a fold change cutoff of 1.5. Categorization of these genes, using Ingenuity Pathway Analysis, MetaCore from Thomson Reuters, and DAVID, revealed genes associated with extracellular matrix remodeling and cell-matrix interactions, amino acid metabolism, and lipid metabolism, as well as immune regulation in the follicular phase tissues. In luteal phase tissues, genes associated with chromatin remodeling, inflammation, angiogenesis, oxidative stress, and immune cell regulation were predominately expressed. Using samples from additional patients' tissues, select genes were confirmed by quantitative real-time PCR; immunohistochemical staining was also done to examine protein levels. This is the first microarray analysis comparing gene expression in endocervix tissues in cycling women. This study identified key genes and molecular pathways that were differentially regulated during the menstrual cycle.

Paracrine activation of WNT/ß-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Identification of FISH biomarkers to detect chromosome abnormalities associated with prostate adenocarcinoma in tumour and field effect environment.

BACKGROUND: To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate. METHODS: Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis. RESULTS: The combination of MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens. CONCLUSIONS: A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review.

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Enhancement of a multianalyte serum biomarker panel to identify lymph node metastases in non-small cell lung cancer with circulating autoantibody biomarkers.

We recently reported the development of a multianalyte serum algorithm to identify nodal status in non-small cell lung cancer (NSCLC) patients facing an anatomic resection with curative intent. This study aims to enhance the overall performance characteristics of this test by adding autoantibody biomarkers identified through immunoproteomic discovery. More specifically, we used sera from 20 NSCLC patients to probe 2-D immunoblots of HCC827 lysates for tumor-associated autoantigens. Relevant differences in immunoreactivity associated with pathological nodal status were then identified via tandem mass spectrometry. Identified autoantigens were then developed into Luminex immunobead assays alongside a series of autoantigen targets relevant to early-disease detection. These assays were then used to measure circulating autoantibody levels in the identical cohort of NSCLC patients used in our original study. This strategy identified 11 autoantigens found primarily in patients with disease progression to the locoregional lymph nodes. Custom Luminex-based "direct-capture" assays (25 total; including autoantibody targets relevant to early-disease detection) were assembled to measure autoantibody levels in sera from 107 NSCLC patients. Multivariate classification algorithms were then used to identify the optimal combination of biomarkers when considered collectively with our original 6-analyte serum panel. The new algorithm resulting from this analysis consists of TNF-α, TNF-RI, MIP-1α and autoantibodies against Ubiquilin-1, hydroxysteroid-(17-ß)-dehydrogenase, and triosephosphate isomerase. The inclusion of autoantibody biomarkers provided a dramatic improvement in the overall test performance characteristics, relative to the original test panel, including an 11% improvement in the classification efficiency.

PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells.

CONTEXT: Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. OBJECTIVE: To determine the function of PLIN2 in leiomyoma cells. DESIGN: Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Forty-one premenopausal women undergoing surgery for fibroids. MAIN OUTCOME MEASURES: Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. RESULTS: PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. CONCLUSIONS: PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

Progesterone receptor integrates the effects of mutated MED12 and altered DNA methylation to stimulate RANKL expression and stem cell proliferation in uterine leiomyoma.

Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.

RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth.

Context: Uterine leiomyomas are the most common type of gynecologic tumor in women. Objective: To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Design: Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Setting: Research laboratory. Patients: Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. Interventions: None. Main Outcome Measure: Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. Results: RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Conclusions: Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases.

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 A X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site.

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None. Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Estrogen receptor ß regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

OBJECTIVE: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. DESIGN: Case-control. SETTING: University research setting. PATIENT(S): Premenopausal women. INTERVENTION(S): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. MAIN OUTCOME MEASURE(S): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) ß. RESULT(S): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERß-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERß based on small interfering RNA knockdown and chromatin immunoprecipitation of ERß followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. CONCLUSION(S): ERß leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.

Alternative splicing of the multidrug resistance protein 1/ATP binding cassette transporter subfamily gene in ovarian cancer creates functional splice variants and is associated with increased expression of the splicing factors PTB and SRp20.

PURPOSE: Overexpression of multidrug resistance protein 1 (MRP1) confers resistance to a range of chemotherapeutic agents in cell lines and could be involved in clinical drug resistance of some tumor types also. We examined MRP1 expression in a small series of untreated human ovarian tumors and matched normal tissues. EXPERIMENTAL DESIGN: We analyzed ten pairs of snap-frozen ovarian tumor and matched normal total ovarian tissues from the same patients for expression of MRP1 by reverse transcription-PCR. Amplified PCR products were sequenced to reveal splicing events of MRP1. MRP1 splice variants were expressed as enhanced green fluorescent fusion proteins in HEK293T cells to demonstrate their localization in the cell and their activity in conferring resistance to doxorubicin. The expression of splicing factors PTB and SRp20 was examined by Western blot. RESULTS: MRP1 was expressed in all 10 of the pairs of specimens. Multiple MRP1 cDNA fragments of various sizes were amplified between exons 10 and 19. Of interest, more MRP1 cDNA fragments were detected in ovarian tumors than in matched normal tissues in 9 of 10 pairs. We identified 10 splicing forms between exons 10 and 19 of the MRP1 gene with exon skipping ranging from 1 to 7. Amplification of the entire coding region of MRP1 from 1 ovarian tumor revealed >20 splice variants. We found whole and partial exon skipping and partial intron inclusion in these splice variants. We expressed 3 of these MRP1 splice variants in HEK293T cells and found that they appeared to localize to the plasma membrane and were functional in conferring resistance to doxorubicin. In addition, we identified a few nucleotide variations in this gene. To understand the basis for increased splice variants in the tumors, we examined splicing factor expression in these tissues. Western blot analysis revealed that two splicing factors, PTB and SRp20, were overexpressed in most ovarian tumors compared with their matched normal ovarian tissues. Importantly, overexpression of both of these splicing factors was associated with the increased number of MRP1 splicing forms in the ovarian tissues. CONCLUSION: The MRP1 gene undergoes alternative splicing at a higher frequency in ovarian tumors than in matched normal tissues. Some of these splice variants confer resistance to doxorubicin. Expression of splicing factors PTB and SRp20 is strongly associated with the alternative splicing of the MRP1 gene.

Amplification and overexpression of topoisomerase IIalpha predict response to anthracycline-based therapy in locally advanced breast cancer.

PURPOSE: The putative association between erbB-2 overexpression and favorable response to anthracyline-based therapy in breast cancer is controversial, and the mechanism unclear. We sought to determine whether coamplification and overexpression of the topoisomerase IIalpha gene, near erbB-2 on chromosome 17, and a known anthracycline target, may underlie the association. EXPERIMENTAL DESIGN: Thirty-five patients who had locally advanced breast cancer (LABC) and who had received neoadjuvant, anthracycline-based therapy were studied. Copy number of topoisomerase IIalpha and erbB-2 was determined by fluorescence in situ hybridization, and expression by immunohistochemistry. RESULTS: Of 8 patients with erbB-2 amplification, 5 had a complete response (CR) or minimal residual disease (MRD), 3 had a partial response (PR), and none had stable (StD) or progressive disease (PD) at the time of mastectomy, versus 3 CR or MRD, 16 PR, and 8 StD or PD for patients without amplification (P = 0.008). In contrast, erbB-2 overexpression was not significantly associated with response (P = 0.114). Of 6 patients with topoisomerase IIalpha amplification, 4 had CR or MRD, 2 PR, and none StD or PD, versus 4 CR or MRD, 17 PR, and 8 StD or PD for patients without amplification (P = 0.034). All of the tumors with topoisomerase IIalpha amplification also had erbB-2 amplification, but not vice versa. Overexpression of topoisomerase IIalpha (9 patients) was also associated with favorable response (P = 0.021). CONCLUSIONS: Coamplification of erbB-2 and topoisomerase IIalpha is significantly associated with favorable local response to anthracycline-based therapy in LABC. The expression data favor a plausible mechanism based on topoisomerase IIalpha biology.

Aberrant EGFR and chromosome 7 associate with outcome in laryngeal cancer.

OBJECTIVES/HYPOTHESIS: Epidermal growth factor receptor (EGFR) over-expression has been reported as a prognostic indicator in laryngeal cancer; however, the association with disease outcome has been inconsistent among studies. Here, we use fluorescence in situ hybridization (FISH) in addition to immunohistochemistry to assess laryngeal squamous cell carcinoma (SCC) to determine whether FISH can better predict patient outcome. STUDY DESIGN: Retrospective study on 59 patients presenting with advanced disease. METHODS: EGFR and chromosome 7 genomic statuses were measured using FISH, and EGFR expression was assessed by immunohistochemistry on formalin-fixed, paraffin-embedded specimens and correlated with outcome in the 59 patients. RESULTS: EGFR expression was marginally associated with outcome, whereas both EGFR and chromosome 7 FISH status were significantly associated with outcome, and the combination of EGFR and chromosome 7 FISH status provided the strongest association of any two combined parameters (P = .0004). Combining EGFR expression with EGFR and chromosome 7 FISH status provided further improvement (P > .0001). CONCLUSIONS: Measurements of EGFR and chromosome 7 FISH status, and to a lesser extent EGFR expression, have potential value in treatment planning for patients with laryngeal SCC.

Uterine Leiomyoma Stem Cells: Linking Progesterone to Growth.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/ß-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

Ovarian steroids, stem cells and uterine leiomyoma: therapeutic implications.

BACKGROUND: Uterine leiomyoma is the most common benign tumor in women and is thought to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a cellular insult. Leiomyomas cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are the most common indication for hysterectomy in the USA. A slow rate of cell proliferation, combined with the production of copious amounts of extracellular matrix, accounts for tumor expansion. A common salient feature of leiomyomas is their responsiveness to steroid hormones, thus providing an opportunity for intervention. METHODS: A comprehensive search of PUBMED was conducted to identify peer-reviewed literature published since 1980 pertinent to the roles of steroid hormones and somatic stem cells in leiomyoma, including literature on therapeutics that target steroid hormone action in leiomyoma. Reviewed articles were restricted to English language only. Studies in both animals and humans were reviewed for the manuscript. RESULTS: Estrogen stimulates the growth of leiomyomas, which are exposed to this hormone not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. The primary action of estrogen, together with its receptor estrogen receptor α (ERα), is likely mediated via induction of progesterone receptor (PR) expression, thereby allowing leiomyoma responsiveness to progesterone. Progesterone has been shown to stimulate the growth of leiomyoma through a set of key genes that regulate both apoptosis and proliferation. Given these findings, aromatase inhibitors and antiprogestins have been developed for the treatment of leiomyoma, but neither treatment results in complete regression of leiomyoma, and tumors recur after treatment is stopped. Recently, distinct cell populations were discovered in leiomyomas; a small population showed stem-progenitor cell properties, and was found to be essential for ovarian steroid-dependent growth of leiomyomas. Interestingly, these stem-progenitor cells were deficient in ERα and PR and instead relied on the strikingly higher levels of these receptors in surrounding differentiated cells to mediate estrogen and progesterone action via paracrine signaling. CONCLUSIONS: It has been well established that estrogen and progesterone are involved in the proliferation and maintenance of uterine leiomyoma, and the majority of medical treatments currently available for leiomyoma work by inhibiting steroid hormone production or action. A pitfall of these therapeutics is that they decrease leiomyoma size, but do not completely eradicate them, and tumors tend to regrow once treatment is stopped. The recent discovery of stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma has the potential to provide the missing link between developing therapeutics that temper leiomyoma growth and those that eradicate them.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.