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author:("Shen, yulong")
1.  CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis  
The Journal of Clinical Investigation  2014;124(4):1672-1684.
The G1 kinase CDK4 is amplified or overexpressed in some human tumors and promotes tumorigenesis by inhibiting known tumor suppressors. Here, we report that CDK4 deficiency markedly accelerated lymphoma development in the Eμ-Myc transgenic mouse model of B lymphoma and that silencing or loss of CDK4 augmented the tumorigenic potential of Myc-driven mouse and human B cell lymphoma in transplant models. Accelerated disease in CDK4-deficient Eμ-Myc transgenic mice was associated with rampant genomic instability that was provoked by dysregulation of a FOXO1/RAG1/RAG2 pathway. Specifically, CDK4 phosphorylated and inactivated FOXO1, which prevented FOXO1-dependent induction of Rag1 and Rag2 transcription. CDK4-deficient Eμ-Myc B cells had high levels of the active form of FOXO1 and elevated RAG1 and RAG2. Furthermore, overexpression of RAG1 and RAG2 accelerated lymphoma development in a transplant model, with RAG1/2-expressing tumors exhibiting hallmarks of genomic instability. Evaluation of human tumor samples revealed that CDK4 expression was markedly suppressed, while FOXO1 expression was elevated, in several subtypes of human non-Hodgkin B cell lymphoma. Collectively, these findings establish a context-specific tumor suppressor function for CDK4 that prevents genomic instability, which contributes to B cell lymphoma. Furthermore, our data suggest that targeting CDK4 may increase the risk for the development and/or progression of lymphoma.
doi:10.1172/JCI63139
PMCID: PMC3973114  PMID: 24614102
2.  Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis 
IL-35 is a member of the IL-12 family of cytokines consisting of IL-12 p35 subunit and IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune suppressive activity. Although IL-35 has been demonstrated to be produced by regulatory T cells, gene expression analysis has revealed that IL-35 is likely to have wider distribution including expression in cancer cells. In this study we have demonstrated that IL-35 is produced in human cancer tissues such as large B cell lymphoma, nasopharyngeal carcinoma and melanoma. In order to determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35 producing plasmacytoma J558 and B16 melanoma cells, and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but stimulates tumorigenesis in both immune competent and Rag1/2 deficient mice. Tumor-derived IL-35 increases CD11b+Gr1+ myeloid cell accumulation in tumor microenvironment, and thereby promotes tumor angiogenesis. In immune competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor antigen specific CD8+ T cell activation, differentiation and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions of IL-35 in promoting tumor growth via enhancing myeloid cell accumulation, tumor angiogenesis and suppression of tumor immunity.
doi:10.4049/jimmunol.1202535
PMCID: PMC3578001  PMID: 23345334
3.  Equivocal and Weakly Positive Hybrid Capture 2 Tests in Women Aged 50 and Older 
American journal of clinical pathology  2013;139(5):10.1309/AJCPARHTB40DTVFV.
The performance of the Hybrid Capture 2 (HC2) test for human papilloma virus (HPV) detection depends on the prevalence of infection. However, the current HC2 manufacturer recommended interpretative algorithm is the same for all women. This test, which may be particularly useful in perimenopausal and postmenopausal women given the morphologic complexity of their Pap tests, could be affected by the overall lower prevalence of HPV infection in this age group. We investigated HC2 equivocal and weakly positive HPV tests in women 50 years and older and the detection of high-grade dysplasia (CIN2+) on their follow-up specimens. All HC2 test data from 1,067 consecutive specimens and 85 additional specimens from women ≥ 50-years-old with equivocal and weakly positive HC2 were analyzed. Follow-up specimens from women with HC2 tests within these ranges were reviewed. No CIN2+ was found on follow-up of 49 cases of women ≥ 50 with equivocal or weakly positive HC2 results. The current HC2 algorithm resulted in “positive” reports in 63% of specimens with initial equivocal HC2 due to retests mostly within the equivocal range. These results suggest that women 50 years and older may benefit from higher HC2 thresholds. The test could also be reported as HC2 values (RLU/CO) to be interpreted in view of risk factors.
doi:10.1309/AJCPARHTB40DTVFV
PMCID: PMC3869231  PMID: 23596112
human papilloma virus; hybrid capture 2; cervical intraepithelial neoplasia; age
4.  Florid diffuse peritoneal deciduosis mimicking carcinomatosis in a primigravida patient: a case report and review of the literature 
A case of a 27 year old G1P0 female with a dichorionic, diamniotic twin pregnancy presenting with premature rupture of membranes found to have omental caking and diffuse yellow-tan peritoneal nodules, clinically suspicious for carcinomatosis. The case work-up showed this to be an example of florid-diffuse peritoneal deciduosis mimicking carcinomatosis which has since resolved 4 months postpartum.
PMCID: PMC3816836  PMID: 24228129
Deciduosis; peritoneal; carcinomatosis; decidua; pregnancy
5.  Polo-like Kinase 3 Functions as a Tumor Suppressor and Is a Negative Regulator of Hypoxia-Inducible Factor-1α under Hypoxic Conditions 
Cancer research  2008;68(11):4077-4085.
Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3−/− mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3−/− mice were large in size, exhibiting enhanced angiogenesis. Plk3−/− mouse embryonic fibroblasts were hypersensitive to the induction of hypoxiainducible factor-1α (HIF-1α) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1α induced by nickel or cobalt ions. Moreover, hypoxia-induced HIF-1α expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1α in mediating the activation of the “survival machinery” in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway.
doi:10.1158/0008-5472.CAN-07-6182
PMCID: PMC3725591  PMID: 18519666
6.  MicroRNA Signature in Thyroid Fine Needle Aspiration Cytology Applied to “Atypia of Undetermined Significance” Cases 
Thyroid  2012;22(1):9-16.
Background
MicroRNA (miR) expression signatures are proposed to be able to differentiate thyroid cancer from benign thyroid lesions. We selected eight miRs (miR-146b, -221, -187, -197, -346, -30d, -138, and -302c) to examine the potential use of miRs to supplement diagnostic cytology in cases designated as “atypia of undetermined significance.”
Methods
: miR expression was measured in thyroid fine needle aspiration (FNA) specimens by quantitative polymerase chain reaction. Gene expression analyses and linear discriminant analysis (LDA) were performed in a training sample set (n=60) to obtain a classification rule to predict FNA cases as benign or malignant. The predictions were cross-validated by comparing with the corresponding histological diagnoses. A validation sample set (n=68) was further tested with the established four-miR LDA classification rule.
Results
A set of four miRs (miR-146b, -221, -187, and -30d) was identified that could differentiate malignant from benign lesions. A four-miR LDA classification rule was obtained and used to predict FNA cases as benign or malignant. For the training sample set, we obtained a diagnostic accuracy of 93.3%, sensitivity of 93.2%, specificity of 93.8%, positive predictive value (PPV) of 0.98, and negative predictive value (NPV) of 0.83. For the validation sample set, we obtained a diagnostic accuracy of 85.3%, sensitivity of 88.9%, specificity of 78.3%, PPV of 0.89, and NPV of 0.78. For the 30 atypia cases in the validation sample set, we obtained a diagnostic accuracy of 73.3%, sensitivity of 63.6%, specificity of 78.9%, PPV of 0.64, and NPV of 0.79. Based on the miR predictions, we classified the atypia cases predicted as “malignant” into “high risk” and those predicted as “benign” into “low risk” categories. While thyroid carcinomas, particularly papillary thyroid carcinomas (PTCs), were relatively enriched in the high-risk category, this particular miR panel is subject to inaccurate results in follicular neoplasias in atypia cases.
Conclusions
We demonstrate that miR amplification from FNA samples is feasible and that the particular four miR profile in this study can identify PTCs. However, further refinement is required for application to FNA cytology of “atypia of undetermined significance” cases due to low accuracy in classifying follicular neoplasias.
doi:10.1089/thy.2011.0081
PMCID: PMC3247703  PMID: 22136206
7.  Dichotomy effects of Akt signaling in breast cancer 
Molecular Cancer  2012;11:61.
Background
The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion.
Results
We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting.
Conclusion
We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.
doi:10.1186/1476-4598-11-61
PMCID: PMC3494580  PMID: 22917467
Activated Akt signaling; Breast epithelia; Epithelial-mesenchymal transition; Motility; Stem-progenitor cells
8.  Epigenetic Repression of RARRES1 Is Mediated by Methylation of a Proximal Promoter and a Loss of CTCF Binding 
PLoS ONE  2012;7(5):e36891.
Background
The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown.
Methodology/Principal Findings
We first compared methylation occurring at the sequences (−664∼+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (−664∼−86), while the downstream element (−85∼+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores.
Conclusion/Significance
This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.
doi:10.1371/journal.pone.0036891
PMCID: PMC3355180  PMID: 22615834
9.  Epigenetic Silencing Mediated Through Activated PI3K/AKT Signaling in Breast Cancer 
Cancer research  2011;71(5):1752-1762.
Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.
doi:10.1158/0008-5472.CAN-10-3573
PMCID: PMC3048165  PMID: 21216892
Epigenetic silencing; H3K27me3; DNA methylation; PI3K/AKT signaling; breast cancer
10.  Cdc25A Regulates Matrix Metalloprotease 1 through Foxo1 and Mediates Metastasis of Breast Cancer Cells ▿ 
Molecular and Cellular Biology  2011;31(16):3457-3471.
Cdc25A is a cell cycle-activating phosphatase, and its overexpression in breast cancers has been shown to correlate with poor prognosis. Most recent studies related to Cdc25A and tumor progression have focused on its role in regulating cell cycle progression. However, less is known about how Cdc25A modulates the metastasis of breast cancer cells. In this study, we revealed that Cdc25A enhances Foxo1 stability by dephosphorylating Cdk2, and Foxo1 was shown to directly regulate transcription of the metastatic factor MMP1. Further studies have shown that overexpression of Cdc25A in breast cancer cells enhances metastasis, whereas its downmodulation inhibits metastasis in mouse models, and the effects of Cdc25A on breast cancer cell metastasis are independent of cell proliferation and apoptosis. Furthermore, we have demonstrated that aberrant Cdc25A in breast cancer patient samples directly correlates with the metastatic phenotype. Further insights into this critical role of Cdc25A in the metastasis of breast cancer cells and the trial of an anti-Cdc25A strategy in mouse models may reveal its therapeutic potential in prevention and treatment of breast cancer cell dissemination.
doi:10.1128/MCB.05523-11
PMCID: PMC3147788  PMID: 21670150
11.  Correction: Germline Stem Cell Gene PIWIL2 Mediates DNA Repair through Relaxation of Chromatin 
PLoS ONE  2011;6(11):10.1371/annotation/2c1c26ac-ee7e-417d-9852-4d2995ad102b.
doi:10.1371/annotation/2c1c26ac-ee7e-417d-9852-4d2995ad102b
PMCID: PMC3228704
12.  Germline Stem Cell Gene PIWIL2 Mediates DNA Repair through Relaxation of Chromatin 
PLoS ONE  2011;6(11):e27154.
DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili-/- MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine–guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.
doi:10.1371/journal.pone.0027154
PMCID: PMC3217960  PMID: 22110608
13.  Prospective Study of Prostate Tumor Angiogenesis and Cancer-Specific Mortality in the Health Professionals Follow-Up Study 
Journal of Clinical Oncology  2009;27(33):5627-5633.
Purpose
Tumor growth requires the development of independent vascular networks that are often primitive in morphology and function. We examined whether microvessel morphology contributes to the considerable biologic heterogeneity of prostate cancer.
Methods
We evaluated microvessel morphology as a predictor of prostate cancer mortality among 572 men in the Health Professionals Follow-Up Study diagnosed with cancer during 1986 to 2000. We immunostained prostatectomy tumor block sections for endothelial marker CD34 and assessed microvessel density, vessel size (area and diameter), and irregularity of vessel lumen using image analysis. Proportional hazards models were used to assess microvessel density and morphology in relation to lethal prostate cancer.
Results
Poorly differentiated tumors exhibited greater microvessel density, greater irregularity of the vessel lumen, and smaller vessels. During 20 years of follow-up, 44 men developed bone metastases or died of cancer. Men with tumors exhibiting the smallest vessel diameter, based on quartiles, were 6.0 times more likely (95% CI, 1.8 to 20.0) to develop lethal prostate cancer. Men with the most irregularly shaped vessels were 17.1 times more likely (95% CI, 2.3 to 128) to develop lethal disease. Adjusting for Gleason grade and prostate-specific antigen levels did not qualitatively change the results. Microvessel density was not linked to cancer-specific mortality after adjusting for clinical factors.
Conclusion
Aggressive tumors form vessels that are primitive in morphology and function, with consequences for metastases. Vascular size and irregularity reflect the angiogenic potential of prostate cancer and may serve as biomarkers to predict prostate cancer mortality several years after diagnosis.
doi:10.1200/JCO.2008.20.8876
PMCID: PMC2792955  PMID: 19858401
14.  Identification of Piwil2-Like (PL2L) Proteins that Promote Tumorigenesis 
PLoS ONE  2010;5(10):e13406.
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G0/1 into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.
doi:10.1371/journal.pone.0013406
PMCID: PMC2958115  PMID: 20975993
15.  Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma 
The role of aberrant methylation of fragile histidine triad (FHIT) promoters in the differentiated thyroid carcinoma (DTC) is not yet clear. Therefore, we investigated the association of the status of FHIT promoter methylation and FHIT protein expression with the clinicopathological progression of DTC, using PCR-based methylation assay and immunohistochemical technique. While no FHIT gene promoter methylation was observed in the matched non-cancerous epithelium (NCE) specimens, 24.6% of DTC samples demonstrated methylation in the FHIT promoter region. The protein expression of FHIT in NCE and DTC was 100.0% and 41.5% (P<0.01), respectively. There was a negative correlation between promoter methylation and protein expression of FHIT gene (P<0.05). Additionally, the methylation status appeared to be significantly associated with the pathological grade, tumor TNM stage, and lymph node metastasis (P<0.05), and FHIT proteins were weakly expressed in only about 20% of DTC with grade II pathological changes, TNM stage III/IV, or lymph node metastasis. Finally, the gender and tumor classification but not age marginally affected the promoter methylation and protein expression of FHIT. Our results suggest that methylation of the promoter region may play a key role in inactivation of FHIT - possibly leading to subsequent carcinogenesis and progression of DTC.
PMCID: PMC2897109  PMID: 20606729
Fragile histidine triad; differentiated thyroid carcinoma; methylation; carcinogenesis; tumor progression
16.  Piwil2 expressed in various stages of cervical neoplasia is a potential complementary marker for p16INK4a 
Generally, cancers may undergo the developmental stages of benign proliferation, precancer and invasive cancer. Identification of biomarkers that are expressed throughout the developmental stages will facilitate detection, prevention and therapy of cancer. Piwil2, a member of AGO/PIWI family of proteins, has been suggested to be associated with tumor development. Here we reported that piwil2 can be detected by immunohistochemistry (IHC) in various stages of human cervical squamous cell carcinomas and adenocarcinomas. Interestingly, piwil2 was also detected in some metaplastic epithelial cells as well as histologically “normal” appearing tissues adjacent to malignant lesions. While all the premalignant and malignant lesions expressed varying levels of piwil2, p16INK4a (p16), a surrogate indicator of high-risk human papillomavirus (HR-HPV) infection, was detected in only 84.62% of the specimens. In Papanicolaou (Pap) test, piwil2 was also detected in atypical glandular cells (AGC), low-grade (LSIL) and high-grade squamous intraepithelial lesions (HSIL), whereas p16 was not always concomitantly detected in the same specimens. The results suggest that piwil2 might play important roles throughout the process of cervical cancer development and have the potential to be used as a complementary marker for p16INK4a. It is worth further study to improve the sensitivity and specificity of current screening methods for cervical cancers.
PMCID: PMC2855633  PMID: 20407605
Piwil2; precancer; tumor development; field cancerization; cervical cancer; p16INK4a
17.  Piwil2 is expressed in various stages of breast cancers and has the potential to be used as a novel biomarker 
Piwil2, a member of AGO/PIWI family of proteins, has been reported to be expressed in precancerous stem cells (pCSCs), tumor cell lines and various types of human cancers. However, the significance of piwil2 expression in breast cancer has not been investigated. In this study, archival formalin-fixed, paraffin-embedded breast cancer specimens at various developmental stages were prepared as tissue microarrays (TMAs) and examined for the expressions of piwil2, estrogen receptor (ER), progesterone receptor (PR) and a cell proliferation marker Ki67 by immu-nohistochemical (IHC) staining and human epidermal growth factor receptor 2 (HER2) by fluorescence in situ hybridization (FISH). The correlation of piwil2 expression with ER, PR and Ki67 were analyzed statistically. The piwil2 was detected in all of breast cancer TMA cores. In contrast, ER, PR, HER2, and Ki67 were detected only in 66.1%, 54.5%, 36.0%, and 47% of the TMA cores, respectively. Piwil2 was expressed in cytoplasm (Cyt), nucleus (N) or both cytoplasm and nucleus (C-N). The N pattern was less observed in breast precancers, whereas all three patterns were observed in invasive and metastatic cancers. While the Cyt pattern was significantly correlated with ER expression (p = 0.002); N pattern was significantly correlated with Ki67 expression (p =0.001). ER and Ki67 expressions were reduced and increased, respectively, with the expression patterns being shifted from Cyt → C-N → N. In conclusion, piwil2 is expressed in various stages of breast cancers and has the potential to be used a novel biomarker.
PMCID: PMC2872741  PMID: 20490325
Piwil2; breast cancer; precancer; estrogen receptor; progesterone receptor; HER2; and Ki67; field cancerization
18.  Breast Cancer-associated Fibroblasts Confer AKT1-mediated Epigenetic Silencing of Cystatin M in Epithelial Cells 
Cancer research  2008;68(24):10257.
The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells co-cultured with cancer-associated fibroblasts (CAFs). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulted from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contract between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Since this two-way interaction is anticipated, the described co-culture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.
doi:10.1158/0008-5472.CAN-08-0288
PMCID: PMC2821873  PMID: 19074894
19.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
doi:10.1016/j.cell.2007.04.034
PMCID: PMC1974845  PMID: 17570480
20.  Reversibility of Aberrant Global DNA and Estrogen Receptor-α Gene Methylation Distinguishes Colorectal Precancer from Cancer 
Alterations in the global methylation of DNA and in specific regulatory genes are two epigenetic alterations found in cancer. However, the significance of epigenetic changes for diagnosis and/or prognosis of colorectal cancer have not been established, although it has been extensively investigated. Recently we have identified a new type of cancer cell called precancerous stem cells (pCSCs) and proposed that cancer may arise from a lengthy development process of tumor initiating cells (TICs) → pCSCs → cancer stem cells (CSCs) → cancer, which is in parallel to histological changes of hyperplasia (TICs) → precancer (pCSCs) → carcinoma (CSCs/cancer cells), accompanied by clonal evolutionary epigenetic and genetic alterations. In this study, we investigated whether aberrant DNA methylation can be used as a biomarker for the differentiation between premalignant and malignant lesions in the colorectum. The profile of global DNA and estrogen receptor (ER)-α gene methylation during cancer development was determined by analysis of 5-methylcytosine (5-MeC) using immunohistochemical (IHC) staining, dot blot analysis or a quantitative gene methylation assay (QGMA). Herein we show that global DNA hypomethylation and ER-α gene hypermethylation are progressively enhanced from hyperplastic polyps (HPs) → adenomatous polyps (APs) → adenomatous carcinoma (AdCa). The aberrant methylation can be completely reversed in APs, but not in AdCa by a nonsteroidal anti-inflammatory drug (NSAID) celecoxib, which is a selective inhibitor of cyclooxygenase-2 (Cox-2), suggesting that the epigenetic alterations between colorectal precancer (AP) and cancer (AdCa) are fundamentally different in response to anti-cancer therapy. In normal colorectal mucosa, while global DNA methylation was not affected by aging, ER-α gene methylation was significantly increased with aging. However, this increase did not reach the level observed in colorectal APs. Taken together, reversibility of aberrant global DNA and ER-α gene methylation distinguishes colorectal precancer from cancer.
PMCID: PMC2491387  PMID: 18830381
Precancer; DNA methylation; colorectal cancer; estrogen receptor-α; nonsteroidal anti-inflammatory drugs; cancer progression; epigenetic; tumor initiation
21.  Precancerous Stem Cells Can Serve As Tumor Vasculogenic Progenitors 
PLoS ONE  2008;3(2):e1652.
Tumor neo-vascularization is critical for tumor growth, invasion and metastasis, which has been considered to be mediated by a mechanism of angiogenesis. However, histopathological studies have suggested that tumor cells might be the progenitor for tumor vasculature. Recently, we have reported that the precancerous stem cells (pCSCs) representing the early stage of developing cancer stem cells (CSCs), have the potential for both benign and malignant differentiation. Therefore, we investigated whether pCSCs serve as progenitors for tumor vasculogenesis. Herein, we report that in the pCSC-derived tumors, most blood vessels were derived from pCSCs. Some pCSCs constitutively expressed vasculogenic receptor VEGFR-2, which can be up-regulated by hypoxia and angiogenesis-promoting cytokines, such as GM-CSF, Flt3 ligand, and IL-13. The pCSCs are much more potent in tumor vasculogenesis than the differentiated tumor monocytic cells (TMCs) from the same tumor, which had comparable or even higher capacity to produce some vascular growth factors, suggesting that the potent tumor vasculogenesis of pCSCs is associated with their intrinsic stem-like property. Consistently tumor vasculogenesis was also observed in human cancers such as cervical cancer and breast cancer and xenograft lymphoma. Our studies indicate that pCSCs can serve as tumor vasculogenic stem/progenitor cells (TVPCs), and may explain why anti-angiogenic cancer therapy trials are facing challenge.
doi:10.1371/journal.pone.0001652
PMCID: PMC2242848  PMID: 18286204
22.  Invasive Micropapillary Carcinoma of the Sigmoid Colon: Distinct Morphology and Aggressive Behavior 
We report a case of invasive micropapillary carcinoma of the sigmoid colon in a 72-year-old female with anemia and abdominal pain. Grossly, the tumor demonstrated a deeply invasive, ulcerated fungating mass. Microscopically, the carcinoma was predominantly composed of micropapillae with reversed cell polarity, abundant neutrophils, and surrounded by clear spaces. Multifocal lymphovascular invasion was present with extensive lymph node metastasis. Immunohistochemically, the carcinoma cells were positive for CDX2, CK20 and monoclonal carcinoembryonic antigen. They were negative for CK7. The stroma-facing surface of the micropapillae was positive for CD10 and villin, confirming the inside-out growth pattern characteristic of micropapillary carcinoma. Work-up for distance metastasis was negative. The patient was alive and well 1.5 years after sigmoidectomy and postoperative chemotherapy.
PMCID: PMC2480571  PMID: 18787620
Adenocarcinoma; micropapillary; invasive; metastasis; colon
23.  Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation 
PLoS ONE  2007;2(3):e293.
Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.
doi:10.1371/journal.pone.0000293
PMCID: PMC1808425  PMID: 17356702

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