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Supplementary Data 1 from Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

Supplementary Data 2 from Identification of Smyd4 as a Potential Tumor Suppressor Gene Involved in Breast Cancer Development

To identify genes involved in breast tumorigenesis, we applied the retroviral LoxP-Cre system to a nontumorigenic mouse mammary epithelial cell line NOG8 to create random chromosome deletion/translocation. We found that the disruption of one allele of Smyd4 (SET and MYND domain containing 4) gene through chromosome translocation led to tumorigenesis. The expression of Smyd4 was markedly decreased in tumor cells. Re-expression of Smyd4 resulted in growth suppression of tumor cells and inhibition of tumor formation in nude mice. Furthermore, the RNA interference–mediated suppression of Smyd4 expression in human MCF10A mammary epithelial cells caused their growth in soft agar. Microarray studies revealed that platelet-derived growth factor receptor α polypeptide (Pdgfr-α) was highly expressed in tumor cells compared with NOG8 cells. Re-expression of Smyd4 significantly reduced the expression of Pdgfr-α in tumor cells. In human breast cancers, reverse transcription-PCR results revealed that Smyd4 expression was totally silenced in 2 of 10 specimens. These findings indicate that Smyd4, as a potential tumor suppressor, plays a critical role in breast carcinogenesis at least partly through inhibiting the expression of Pdgfr-α, and could be a novel target for improving treatment of breast cancer. [Cancer Res 2009;69(9):4067–72]

Plexcitons are polaritonic modes that result from the strong coupling between excitons and plasmons. Here, we consider plexcitons emerging from the interaction of excitons in an organic molecular layer with surface plasmons in a metallic film. We predict the emergence of Dirac cones in the two-dimensional band-structure of plexcitons due to the inherent alignment of the excitonic transitions in the organic layer. An external magnetic field opens a gap between the Dirac cones if the plexciton system is interfaced with a magneto-optical layer. The resulting energy gap becomes populated with topologically protected one-way modes, which travel at the interface of this plexcitonic system. Our theoretical proposal suggests that plexcitons are a convenient and simple platform for the exploration of exotic phases of matter and for the control of energy flow at the nanoscale., United States. Department of Energy. Office of Basic Energy Sciences (Award DESC0001088), Solid-State Solar-Thermal Energy Conversion Center (Award DE-SC0001299)

3D integration utilizing TSV (Through-Silicon Via) is a promising technology to achieve high performance 3DIC (three-dimentional integrated circuit), not only because it exponentially increases connection efficiency between dies, but also because it can be practically integrated into current IC manufacture processes with minimal impact on both FEOL and BEOL design and processing. During construction of TSV, CMP (Chemical-Mechanical Polishing) is required to reveal the vias and finish the wafer surface for future processing. Reported in this paper is our recent effort in developing a series of slurries for TSV front-side polishing by close collaboration in device design, slurry formulation and process fine-tuning. Specifically, high through-put and well performed Cu slurries, along with high SiO2/Si3N4 selectivity barrier slurries, are introduced. These slurries are tested on laboratory-scale instruments as well as foundry CMP tools. Polishing results from both blanket and pattern wafers are discussed.

III-IV-V heterovalent alloys have the potential to satisfy the need for infrared bandgap materials that also have lattice constants near GaAs. In this work, significant room temperature photoluminescence is reported for the first time in high quality III-IV-V alloys grown by metalorganic chemical vapor deposition. Pronounced phase separation, a characteristic suspected to quench luminescence in the alloys in the past, was successfully inhibited by a modified growth process. Small scale composition fluctuations were observed in the alloys; higher growth temperatures resulted in fluctuations with a striated morphology, while lower growth temperatures resulted in fluctuations with a speckled morphology. The composition fluctuations cause bandgap narrowing in the alloys—measurements of various compositions of (GaAs)1-x(Ge2)x alloys reveal a maximum energy transition of 0.8 eV under 20% Ge composition rather than a continuously increasing transition with the decreasing Ge composition. Additionally, luminescenc...

Nuclear receptor coactivator [peroxisome proliferator-activated receptor-binding protein (PBP)/mediator subunit 1 (MED1)] is a critical component of the mediator transcription complex. Disruption of this gene in the mouse results in embryonic lethality. Using the PBP/MED1 liver conditional null (PBP/MED1(DeltaLiv)) mice, we reported that PBP/MED1 is essential for liver regeneration and the peroxisome proliferator-activated receptor alpha ligand Wy-14,643-induced receptor-mediated hepatocarcinogenesis. We now examined the role of PBP/MED1 in genotoxic chemical carcinogen diethylnitrosamine (DEN)-induced and phenobarbital-promoted hepatocarcinogenesis. The carcinogenic process was initiated by a single intraperitoneal injection of DEN at 14 days of age and initiated cells were promoted with phenobarbital (PB) (0.05%) in drinking water. PBP/MED1(DeltaLiv) mice, killed at 1, 4 and 12 weeks, revealed a striking proliferative response of few residual PBP/MED1-positive hepatocytes that escaped Cre-mediated deletion of PBP/MED1 gene. No proliferative expansion of PBP/MED1 null hepatocytes was noted in the PBP/MED1(DeltaLiv) mouse livers. Multiple hepatocellular carcinomas (HCCs) developed in the DEN-initiated PBP/MED1(fl/fl) and PBP/MED1(DeltaLiv) mice, 1 year after the PB promotion. Of interest is that all HCC developing in PBP/MED1(DeltaLiv) mice were PBP/MED1 positive. None of the tumors was PBP/MED1 negative implying that hepatocytes deficient in PBP/MED1 are not susceptible to neoplastic conversion. HCC that developed in PBP/MED1(DeltaLiv) mouse livers were transplantable in athymic nude mice and these maintained PBP/MED1(fl/fl) genotype. PBP/MED1(fl/fl) HCC cell line derived from these tumors expressed PBP/MED1 and deletion of PBP/MED1(fl/fl) allele by adeno-Cre injection into tumors caused necrosis of tumor cells. These results indicate that PBP/MED1 is essential for the development of HCC in the mouse.

PRIP (peroxisome proliferator-activator receptor interacting protein) is a nuclear receptor coactivator required for mammary gland development. To understand the function of PRIP in breast tumorigenesis, we established a mammary tumor cell line with the PRIPLoxp/Loxp genotype. By knocking out the PRIP gene in the tumor cell line, we demonstrated that PRIP deficiency led to inhibited tumor formation without affecting tumor cell proliferation. The PRIP deficiency was associated with decreased cell invasion and migration capabilities. We found that PRIP deficiency substantially reduced FOS gene expression. A chromatin immunoprecipitation assay revealed that PRIP was recruited to the FOS promoter. In addition, we demonstrated that PRIP also directly up-regulated the FOS gene expression in human breast cancer cells. Promoter analysis showed that PRIP acted through serum-responsive factor to regulate FOS gene expression. Finally, by re-expressing the FOS gene, we confirmed that the inhibited tumor formation of PRIP-deficient tumor cells was due to reduced expression of the FOS gene.

To identify genes involved in breast tumorigenesis, we applied the retroviral LoxP-Cre system to a nontumorigenic mouse mammary epithelial cell line NOG8 to create random chromosome deletion/translocation. We found that the disruption of one allele of Smyd4 (SET and MYND domain containing 4) gene through chromosome translocation led to tumorigenesis. The expression of Smyd4 was markedly decreased in tumor cells. Re-expression of Smyd4 resulted in growth suppression of tumor cells and inhibition of tumor formation in nude mice. Furthermore, the RNA interference–mediated suppression of Smyd4 expression in human MCF10A mammary epithelial cells caused their growth in soft agar. Microarray studies revealed that platelet-derived growth factor receptor α polypeptide (Pdgfr-α) was highly expressed in tumor cells compared with NOG8 cells. Re-expression of Smyd4 significantly reduced the expression of Pdgfr-α in tumor cells. In human breast cancers, reverse transcription-PCR results revealed that Smyd4 expression was totally silenced in 2 of 10 specimens. These findings indicate that Smyd4, as a potential tumor suppressor, plays a critical role in breast carcinogenesis at least partly through inhibiting the expression of Pdgfr-α, and could be a novel target for improving treatment of breast cancer. [Cancer Res 2009;69(9):4067–72]

To investigate the role of nuclear receptor coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) in mammary gland development, we generated a conditional null mutation of PRIP in mammary glands. In PRIP-deficient mammary glands, the elongation of ducts during puberty was not affected, but the numbers of ductal branches were decreased, a condition that persisted long after puberty, indicating that the potential of ductal branching was impaired. During pregnancy, PRIP-deficient mammary glands exhibited decreased alveolar density. The lactating PRIP-deficient glands contained scant lobuloalveoli with many adipocytes, whereas the wild type glands were composed of virtually no adipocytes but mostly lobuloalveoli. As a result, PRIP mammary-deficient glands could not produce enough milk to nurse all the pups during lactation. The ductal branching of mammary glands in response to estrogen treatment was attenuated in PRIP mutant glands. Whereas the proliferation index was similar between wild type and PRIP-deficient glands, increased apoptosis was observed in PRIP-deficient glands. PRIP-deficient glands expressed increased amphiregulin, transforming growth factor-alpha, and betacellulin mRNA as compared with wild type glands. The differentiated function of PRIP-deficient mammary epithelial cells was largely intact, as evidenced by the expression of abundant beta-casein, whey acidic protein (WAP), and WDNM1 mRNA. We conclude that PRIP is important for normal mammary gland development.

ASH2L is a component of MLL complexes that confer H3K4 trimethylation. The ASH2L gene is located at 8q11–12, which is often amplified in breast cancers. We found that increased ASH2L expression, which can result from gene amplification, is often correlated with increased ERα expression in both breast cancer cell lines and primary breast cancers. Forced expression of ASH2L induced ERα expression in mammary epithelial cells, whereas depletion of ASH2L suppressed ERα expression in breast cancer cells. To understand the mechanism by which ASH2L regulates ERα expression, we identified GATA3 as the binding protein of ASH2L. ASH2L was shown to potentiate the transcriptional activity of GATA3. ASH2L was recruited to the enhancer of the ERα gene through GATA3 to promote ERα transcription. This study established that ASH2L enhances ERα expression as a coactivator of GATA3 in breast cancers.

The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors. In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2/AIB3/RAP250/NRC/TRBP) as bait in a yeast two-hybrid screen. Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant G XX G XX I segment found in K-homology motifs of many RNA-binding proteins. The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb. PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta. Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus. PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773–927. PIMT binds S -adenosyl- l -methionine, the methyl donor for methyltransfer reaction, and it also binds RNA, suggesting that it is a putative RNA methyltransferase. PIMT enhances the transcriptional activity of peroxisome proliferator-activated receptor γ and retinoid-X-receptor α, which is further stimulated by coexpression of PRIP, implying that PIMT is a component of nuclear receptor signal transduction apparatus acting through PRIP. Definitive identification of the specific substrate of PIMT and the role of this RNA-binding protein in transcriptional regulation remain to be determined.

PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.

Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a ∼3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non-tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re-expression of Fat4 in Fat4-deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non-tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene. © 2008 Wiley-Liss, Inc.

Chromosome Deletions: Where Less Says More The ability to generate large chromosomal deletions and assay the phenotype in cells can provide valuable information regarding genes located within the d...

To identify estrogen-responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERalpha interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERalpha was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells.

Breast cancer amplified sequence 2 (BCAS2) was initially identified as a gene that was overexpressed and amplified in some breast cancer cell lines. It was later found to be a component of the spliceosome. Here, we identified BCAS2 as an estrogen receptor (ER) alpha interacting protein by yeast two-hybrid screening. In addition to ER alpha, BCAS2 also interacted with ER beta, TR beta, PR, and PPAR gamma in a ligand-independent way. Transient transfection assays revealed that overexpression of BCAS2 enhanced while inhibition of BCAS2 expression attenuated the estrogen receptor-mediated transcription. BCAS2 potentiated the activation function-2 (AF-2) activity of ER alpha but had no effect on the AF-1 activity. This study suggested that BCAS2 might play an important role in breast cancer development by increasing the estrogen receptor's function.

To understand the mechanism by which estrogen receptor (ER) activates transcription in a tissue specific fashion, we isolated ERalpha binding protein (ERBP) by performing yeast two-hybrid screening with human mammary gland cDNA library. ERBP is a nuclear protein and its mRNA is ubiquitously expressed. The in vitro interaction of ERBP with ERalpha was demonstrated by GST pull-down assay and this interaction was enhanced by estrogen. In addition, ERBP also bound to PPARgamma, RXRalpha, and ERbeta. ERBP interacted with the DNA binding domain and the hinge region of ERalpha. There are two ERalpha binding regions on ERBP. The binding of ERBP region at C-terminus to ERalpha is increased by estrogen while the binding of ERBP region at N-terminus is not affected by estrogen. The interaction of ERBP with ERalpha was further confirmed in vivo by immunoprecipitation. Transient transfection experiment demonstrated that ERBP enhanced the transcriptional activity of ERalpha.

Peroxisome proliferators are postulated to elicit predictable pleiotropic responses in the liver by activating a peroxisome proliferator-activated receptor (PPAR). PPARs from mouse liver (mPPAR), rat liver (rPPAR), and Xenopus liver (xPPAR gamma) have been cloned recently. We now report the cloning of a new member from mouse liver which we designate mPPAR gamma. mPPAR gamma cDNA contained an open reading frame encoding a 475-amino acid protein exhibiting 75% amino acid similarity to xPPAR gamma, while it showed only 55% identity with mPPAR. The ligand-binding and DNA-binding domains are best conserved between mPPAR gamma, mPPAR, and xPPAR gamma. Like rPPAR, mPPAR gamma is able to impart peroxisome proliferator responsiveness to the promoter of peroxisomal bifunctional gene, which encodes the second enzyme of the peroxisomal fatty acid beta-oxidation system. Northern blot analysis revealed high expression of mPPAR gamma gene in mouse liver, kidney, and heart and low expression in the lung, testis, brain, skeletal muscle, and spleen. In mice treated with ciprofibrate, a peroxisome proliferator, a 2-fold increase in mPPAR gamma mRNA was observed in the liver and kidney. The presence of two PPARs in the mouse liver suggests the possibility of multiple signaling pathways for the peroxisome proliferator-induced pleiotropic responses.

The speed of solid-state electronic devices, determined by the temporal dynamics of charge carriers, could potentially reach unprecedented petahertz frequencies through direct manipulation by optical fields, consisting in a million-fold increase from state-of-the-art technology. In graphene, charge carrier manipulation is facilitated by exceptionally strong coupling to optical fields, from which stems an important back-action of photoexcited carriers. Here we investigate the instantaneous response of graphene to ultrafast optical fields, elucidating the role of hot carriers on sub-100 fs timescales. The measured nonlinear response and its dependence on interaction time and field polarization reveal the back-action of hot carriers over timescales commensurate with the optical field. An intuitive picture is given for the carrier trajectories in response to the optical-field polarization state. We note that the peculiar interplay between optical fields and charge carriers in graphene may also apply to surface states in topological insulators with similar Dirac cone dispersion relations., Graphene possesses a nonlinear optical response arising from its electronic dispersion. Here, the authors measure the response of graphene to an ultrafast optical field and provide an explanation of the quantum dynamics of Dirac carriers mediating the material’s nonlinear response.