Synthesis of Weakly Confined, Cube-Shaped, and Monodisperse Cadmium Chalcogenide Nanocrystals with Unexpected Photophysical Properties.

Zinc-blende CdSe, CdS, and CdSe/CdS core/shell nanocrystals with a structure-matched shape (cube-shaped, edge length ≤30 nm) are synthesized via a universal scheme. With the edge length up to five times larger than exciton diameter of the bulk semiconductors, the nanocrystals exhibit novel properties in the weakly confined size regime, such as near-unity single exciton and biexciton photoluminescence (PL) quantum yields, single-nanocrystal PL nonblinking, mixed PL decay dynamics of exciton and free carriers with sub-microsecond monoexponential decay lifetime, and stable yet extremely narrow PL full width at half maximum (FWHM < 0.1 meV) at 1.8 K. Their monodisperse edge length, shape, and facet structure enable demonstration of unexpected yet size-dependent PL properties at room temperature, including unusually broad and abnormally size-dependent PL FWHM (∼100 meV), nonmonotonic size dependence of PL peak energy, and dual-peak single-exciton PL. Calculations suggest that these unusual properties should be originated from the band-edge electron/hole states of the dynamic-exciton, whose exciton binding energy is too small to hold the photogenerated electron-hole pair as a bonded Wannier exciton in a weakly confined nanocrystal.

Association of human breast cancer CD44-/CD24- cells with delayed distant metastasis.

Tumor metastasis remains the main cause of breast cancer-related deaths, especially delayed breast cancer distant metastasis. The current study assessed the frequency of CD44-/CD24- breast cancer cells in 576 tissue specimens for associations with clinicopathological features and metastasis and investigated the underlying molecular mechanisms. The results indicated that higher frequency (≥19.5%) of CD44-/CD24- cells was associated with delayed postoperative breast cancer metastasis. Furthermore, CD44-/CD24-triple negative breast cancer (TNBC) cells spontaneously converted into CD44+/CD24-cancer stem cells (CSCs) with properties similar to CD44+/CD24-CSCs from primary human breast cancer cells and parental TNBC cells in terms of stemness marker expression, self-renewal, differentiation, tumorigenicity, and lung metastasis in vitro and in NOD/SCID mice. RNA sequencing identified several differentially expressed genes (DEGs) in newly converted CSCs and RHBDL2, one of the DEGs, expression was upregulated. More importantly, RHBDL2 silencing inhibited the YAP1/USP31/NF-κB signaling and attenuated spontaneous CD44-/CD24- cell conversion into CSCs and their mammosphere formation. These findings suggest that the frequency of CD44-/CD24- tumor cells and RHBDL2 may be valuable for prognosis of delayed breast cancer metastasis, particularly for TNBC.

Sequence-dependent recruitment of SRSF1 and SRSF7 to intronless lncRNA NKILA promotes nuclear export via the TREX/TAP pathway.

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.

XAB2 depletion induces intron retention in POLR2A to impair global transcription and promote cellular senescence.

XAB2 is a multi-functional protein participating processes including transcription, splicing, DNA repair and mRNA export. Here, we report POLR2A, the largest catalytic subunit of RNA polymerase II, as a major target gene down-regulated after XAB2 depletion. XAB2 depletion led to severe splicing defects of POLR2A with significant intron retention. Such defects resulted in substantial loss of POLR2A at RNA and protein levels, which further impaired global transcription. Treatment of splicing inhibitor madrasin induced similar reduction of POLR2A. Screen using TMT-based quantitative proteomics identified several proteins involved in mRNA surveillance including Dom34 with elevated expression. Inhibition of translation or depletion of Dom34 rescued the expression of POLR2A by stabilizing its mRNA. Immuno-precipitation further confirmed that XAB2 associated with spliceosome components important to POLR2A expression. Domain mapping revealed that TPR motifs 2-4 and 11 of XAB2 were critical for POLR2A expression by interacting with SNW1. Finally, we showed POLR2A mediated cell senescence caused by XAB2 deficiency. Depletion of XAB2 or POLR2A induced cell senescence by up-regulation of p53 and p21, re-expression of POLR2A after XAB2 depletion alleviated cellular senescence. These data together support that XAB2 serves as a guardian of POLR2A expression to ensure global gene expression and antagonize cell senescence.

Nuclear retention element recruits U1 snRNP components to restrain spliced lncRNAs in the nucleus.

In contrast to cytoplasmic localization of spliced mRNAs, many spliced lncRNAs are localized in the nucleus. To investigate the mechanism, we used lncRNA MEG3 as a reporter and mapped a potent nuclear retention element (NRE), deletion of this element led to striking export of MEG3 from the nucleus to the cytoplasm. Insertion of the NRE resulted in nuclear retention of spliced lncRNA as well as spliced mRNA. We further purified RNP assembled on the NRE in vitro and identified the proteins by mass spectrometry. Screen using siRNA revealed depletion of U1 snRNP components SNRPA, SNRNP70 or SNRPD2 caused significant cytoplasmic localization of MEG3 reporter transcripts. Co-knockdown these factors in HFF1 cells resulted in an increased cytoplasmic distribution of endogenous lncRNAs. Together, these data support a model that U1 snRNP components restrain spliced lncRNAs in the nucleus via the interaction with nuclear retention element.

Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive breast cancer.

BACKGROUND: ErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically by membrane physical properties. The present study aims to investigate the influence of membrane cholesterol contents on ErbB2 status and cellular responses to its tyrosine kinase inhibitors. METHODS: The cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with altered membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model. RESULTS: Membrane cholesterol contents positively correlated with cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of cell membranes. Reduction in cholesterol abundance assisted the internalization and degradation of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory effects of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin also synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive breast cancer xenografts. CONCLUSION: The cell surface distribution of ErbB2 was closely regulated by membrane physical properties governed by cholesterol contents. The cholesterol-lowering medications can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.

Aurora-A Kinase: A Potent Oncogene and Target for Cancer Therapy.

The Aurora kinase family is comprised of three serine/threonine kinases, Aurora-A, Aurora-B, and Aurora-C. Among these, Aurora-A and Aurora-B play central roles in mitosis, whereas Aurora-C executes unique roles in meiosis. Overexpression or gene amplification of Aurora kinases has been reported in a broad range of human malignancies, pointing to their role as potent oncogenes in tumorigenesis. Aurora kinases therefore represent promising targets for anticancer therapeutics. A number of Aurora kinase inhibitors (AKIs) have been generated; some of which are currently undergoing clinical evaluation. Recent studies have unveiled novel unexpected functions of Aurora kinases during cancer development and the mechanisms underlying the anticancer actions of AKIs. In this review, we discuss the most recent advances in Aurora-A kinase research and targeted cancer therapy, focusing on the oncogenic roles and signaling pathways of Aurora-A kinases in promoting tumorigenesis, the recent preclinical and clinical AKI data, and potential alternative routes for Aurora-A kinase inhibition.

HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/-KTS isoforms.

WT1 + KTS and -KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/-KTS isoforms were generated by alternative usage of 2 adjacent 5' splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/-KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/-KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of -KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous -KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous -KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/-KTS isoforms.

Expression and clinicopathological significance of FSIP1 in breast cancer.

AIM: To investigate the clinicopathological significance of the expression of fibrous sheath interacting protein 1 (FSIP1) in breast cancer, serum samples, and wound fluid from patients with breast cancer. METHODS: Wound fluid and serum samples from female patients with primary breast cancer, recurrent and metastatic breast cancer, and benign tumors were analyzed for FSIP1 expression using ELISA. 286 paraffin-embedded surgical specimens from breast cancer patients with at least 5 years of follow-up were included for FSIP1 expression assay using immunohistochemistry. RESULTS: Expression of FSIP1 protein was significantly higher in breast cancer tissues compared to tumor-adjacent tissues (p = 0.001). Strong correlation was observed between FSIP1 expression and human epidermal growth factor receptor 2 (Her-2) or Ki67 expression in breast cancer (p = 0.027 and 0.002, respectively). Similarly, serum level of FSIP1 was higher in patients with recurrent and metastatic breast cancer compared to that of primary breast cancer (7, 713 ± 3, 065 vs. 4, 713 ± 3, 065 pg/ml, p = 0.003). Finally, patients with high FSIP1 expression showed a worse post-operative disease-specific survival (p = 0.024). CONCLUSION: FSIP1 may play an important role in the tumorigenesis and invasion of breast cancer and is a potential biomarker for breast cancer diagnosis or prognosis.

Polymorphisms in predicted microRNA-binding sites in integrin genes and breast cancer: ITGB4 as prognostic marker.

Integrins control the cell attachment to the extracellular matrix and play an important role in mediating cell proliferation, migration and survival. A number of important cancer-associated integrin genes can be regulated by microRNAs (miRNAs) that bind to their target sites in the 3' untranslated regions. We examined the effect of single-nucleotide polymorphisms (SNPs) in predicted miRNA target sites of six integrin genes (ITGA3, ITGA6, ITGAv, ITGB3, ITGB4 and ITGB5) on breast cancer (BC) risk and clinical outcome. Six SNPs were genotyped in 749 Swedish incident BC cases with detailed clinical data and up to 15 years of follow-up together with 1493 matched controls. We evaluated associations between genotypes and BC risk and clinical tumour characteristics. Survival probabilities were compared between different subgroups. As a novel finding, several SNPs seemed to associate with the hormone receptor status. The strongest association was observed between the A allele of the SNP rs743554 in the ITGB4 gene and oestrogen receptor-negative tumours [odds ratio 2.09, 95% confidence intervals (CIs) 1.19-3.67]. The same SNP was associated with survival. The A allele carriers had a worse survival compared with the wild-type genotype carriers (hazard ratio 2.11, 95% CIs 1.21-3.68). The poor survival was significantly associated with the aggressive tumour characteristics: high grade, lymph node metastasis and high stage. None of the SNPs was significantly associated with BC risk. As the ITGB4 SNP seems to influence tumour aggressiveness and survival, it may have prognostic value in the clinic.

PAI-1 -675 4G/5G polymorphism as a prognostic biomarker in breast cancer.

Extracellular matrix degradation, mediated by the urokinase plasminogen activation (uPA) system, is a critical step in tumor invasion and metastasis. High tumor levels of uPA and its inhibitor PAI-1 have been correlated with poor prognosis in breast cancer. We examined whether genetic variation in the genes of the uPA system affect breast cancer susceptibility and prognosis. We genotyped eight potentially functional single nucleotide polymorphisms (SNPs) in six genes of the uPA system in 959 Swedish breast cancer patients with detailed clinical data and up to 15 years of follow-up together with 952 matched controls. We used the unconditional logistic regression models to evaluate the associations between genotypes and breast cancer risk and tumor characteristics. The Kaplan-Meier method was used to estimate the survival probabilities; the log-rank test was used to test differences between subgroups. None of the SNPs conferred an increased breast cancer risk, but correlation with some traditional prognostic factors was observed for several SNPs. Most importantly, we identified the -675 4G/5G SNP in the PAI-1 gene as a promising prognostic biomarker for breast cancer. Compared to the 4G/4G and 4G/5G genotypes 5G/5G homozygosity correlated significantly with worse survival (RR 2.04, 95% CI 1.45-2.86, P<0.001), especially in patients with more aggressive tumors. 5G/5G homozygotes were also the group with worse survival among lymph node negative cases. Our finding suggests that genotyping PAI-1 -675 4G/5G may help in clinical prognosis of breast cancer.

Promoter polymorphisms in matrix metalloproteinases and their inhibitors: few associations with breast cancer susceptibility and progression.

The importance of matrix metalloproteinases and their inhibitors in tumor progression is well documented. We wanted to investigate if single nucleotide polymorphisms (SNPs) in the promoter regions of these genes are associated with susceptibility to or progression of breast cancer. In this, so far largest case-control study, we genotyped eight SNPs in the MMP1, MMP2, MMP3, MMP9, MMP13, RECK and TIMP3 genes in a well-characterized breast cancer series of 959 cases and 952 controls from Sweden. Even though we did not correct for multiple comparisons, only a few associations were noted. We observed a moderately increased risk for the TT homozygotes of the MMP9-1562 C/T SNP (OR 1.88, 95% CI 0.97-3.63) and for the C allele carriers of the TIMP3-1296 T/C SNP (OR 1.25, 95% CI 1.05-1.50). In the survival analysis, only the TC heterozygotes of the RECK-420 T/C SNP showed a better survival compared to the TT homozygotes (P = 0.02 in all cases and P = 0.03 in lymph node negative cases). None of the other SNPs conferred an increased breast cancer risk, nor did they correlate with survival. A combination of the -585 TT homozygosity in the RECK gene and the -1296 TT homozygosity in the TIMP3 gene correlated with estrogen and progesterone receptor status (OR 1.81, 95% CI 1.03-3.21 and OR 2.10, 95% CI 1.18-3.86, respectively), and a combination of the -1306 TT homozygosity in the MMP2 gene and the -1562 CC homozygosity in the MMP9 gene with progesterone receptor status (OR 2.34, 95% CI 1.08-5.08). Although our study suggests some correlations between the studied SNPs and the progression of breast cancer, the rarity of the risk genotypes limits their usefulness in the clinic.

Phenotypic consequences of branch point substitutions.

The branch point sequence (BPS) is a conserved splicing signal important for spliceosome assembly and lariat intron formation. BPS mutations may result in aberrant pre-mRNA splicing and genetic disorders, but their phenotypic consequences have been difficult to predict, largely due to a highly degenerate nature of the BPS consensus. Here, we have examined the splicing pattern of nine reporter pre-mRNAs that have previously been shown to give rise to human hereditary diseases as a result of single-nucleotide substitutions in the predicted BPS. Increased exon skipping and intron retention observed in vivo were recapitulated for each mutated pre-mRNA, but the reproducibility of cryptic splice site activation was lower. BP mutations in reporter pre-mRNAs frequently induced aberrant 3' splice sites and also activated a cryptic 5' splice site. Systematic mutagenesis of BP adenosines showed that in most pre-mRNAs, the expression of canonical transcripts was lower for BP transitions than BP transversions. Differential splicing outcome for transitions vs. transversions was abrogated or reduced if introns were truncated to 200 nt or less, suggesting that the nature of the BP residue is less critical for interactions across very short introns. Together, these results improve prediction of phenotypic consequences of point mutations upstream of splice acceptor sites and suggest that the overrepresentation of disease-causing adenosine-to-guanosine BP substitutions observed in Mendelian disorders is due to more profound defects of gene expression at the level of pre-mRNA splicing.

CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk.

Mutations and diminished expression of the E-cadherin gene (CDH1) have been identified in a number of epithelial malignancies. Although somatic CDH1 mutations were detected in lobular breast cancer with a frequency ranging from 10-56%, CDH1 alterations in more frequent ductal tumors appear to be rare. Here we have analyzed the coding region of CDH1 for mutations using denaturing high performance liquid chromatography and found 4 mutations in 83 ductal carcinomas (5%) and 3 mutations in 25 lobular carcinomas (12%). The germline of 13 patients with familial lobular tumors was also analyzed for mutations, but none were detected. In a case-control study, we also tested whether a variant adenine allele in the promoter polymorphism -161C-->A with a putative influence on the transcriptional activity of CDH1 in vitro confers any detectable risk of breast cancer. No significant difference in the allelic frequency between patients with breast cancer (326/1,152, 28.3%) and controls (190/696, 27.3%, p > 0.05; relative risk 1.05, 95% confidence interval 0.85-1.30) was found. A novel promoter polymorphism was identified at position -152, but the frequency of the variant cytosine allele was also similar in patients with breast cancer and controls (0.71% vs. 0.21%, p = 0.23). Transient transfection experiments using reporter constructs containing the nucleotide substitutions -161C/-152C and -161A/-152T showed only a slight decrease in the transcription activity compared to the wild-type construct. These results do not support CDH1 as a prominent low-penetrance cancer susceptibility gene, but indicate that CDH1 mutations contribute to the progression of both lobular and ductal tumors.