Cryptococcus neoformans with a Mutation in the Tetratricopeptide Repeat-Containing Gene, CCN1, Causes Subcutaneous Lesions but Fails To Cause Systemic Infection

We studied a Cryptococcus neoformans strain that caused feline chronic nasal granuloma without disseminated disease. This strain, B-4551, grows at temperatures up to 35°C and fails to cause systemic infection in mice. Many cells of B-4551 formed short hyphal elements in feline nasal tissue and occasionally at 35°C in vitro. A complementation and sequence analysis revealed that the temperature-sensitive (Ts) phenotype of B-4551 was due to deletion of a lysine residue in the cryptococcal CCN1 gene. B-4551 complemented with the wild type CCN1 gene grew at 37°C and caused fatal systemic infection in mice. The CCN1 gene encodes a protein containing 16 copies of a tetratricopeptide repeat. CCN1 is homologous to the Saccharomyces cerevisiae CLF1 gene, which is required for pre-mRNA splicing, cell cycle progression, and DNA replication, and to the Drosophila melanogaster crn gene, which is involved in neurogenesis. CLF1 complemented the Ts phenotype of B-4551. CCN1, however, failed to rescue the clf1 mutant in S. cerevisiae. These results indicate that the Ccn1p may not be as functionally diverse as Clf1p in yeast

Constitutive activation of STAT3 signaling regulates hTERT and promotes stem cell-like traits in human breast cancer cells.

Mounting clinical data suggest that high telomerase activity is tightly associated with cancer progression and poor outcomes. Constitutively activated STAT3 is found in ∼60% of human malignancies and shows a dismal prognosis. We previously reported that activated STAT3 promoted epithelial-mesenchymal transition (EMT) and cancer stem cell phenotype in human breast cancer. However, little is known how STAT3 is regulated in the cancer stem cell and by which mechanisms STAT3 contributes to poor prognosis in aggressive breast cancer. Here we demonstrate that STAT3 physically interacts with CD44 and NF-kB and activates the catalytic subunit of telomerase (hTERT) in human breast cancer stem cells. STAT3 plays a role as a signal transducing molecule between CD44 and NF-kB. In addition to functioning as a catalytic subunit of telomerase, hTERT has been reported to function as a transcription co-factor which drives EMT and cancer stem cell phenotype in human cancer. We observed that activated hTERT increases CD44 (+) subpopulation, whereas targeted knock-down of hTERT abolished cancer stem cell phenotype. Targeted STAT3 knock-down cells also down-regulated hTERT and decreased CD44 subpopulation. Finally, CD44 knock-down resulted in the abrogation of cancer stem cell phenotype and concurrent down-regulation of pSTAT3 and hTERT. Our study delineates the signaling pathway where STAT3 functions as a modulator for CD44 and hTERT, promoting a cancer stem cell phenotype. The constitutive activation of STAT3 signaling that leads to regulation of hTERT pathway may provide novel therapeutic targets for human breast cancer stem cells

Propagation of Saccharomyces cerevisiae [PSI(+)] Prion Is Impaired by Factors That Regulate Hsp70 Substrate Binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90

Transcriptional silencing of hTERT suppressed the tumorigenecity and invasiveness <i>in vitro</i>.

<p>A: Tumorosphere formation assay. When hTERT was knocked-down, the tumorosphere formation was inhibited <i>in vitro</i>. hTERT knock-down cells were subjected to three dimensional culture condition and were examined for tumor sphere formation after 5 days. B: Quantitative representation of tumorospheres formed in MCF7-HER2 and hTERT knock-down cells. C: Boyden chamber assay. Cell invasiveness was examined by employing Boyden chamber assay. MCF7-HER2 and hTERT knock-down cells were subjected to Boyden chamber assay. Again, less cells invaded the filters when hTERT expression was silenced. D: The cell invasion assay was quantitatively measured in graphic representation.</p

Molecular Analysis of CPRα, a MATα-Specific Pheromone Receptor Gene of Cryptococcus neoformans

The putative Cryptococcus neoformans pheromone receptor gene CPRα was isolated and studied for its role in mating and filamentation. CPRα is MATα specific and located adjacent to STE12α at the MATα locus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of the CPRα gene exhibited drastic reductions in mating efficiency but were not completely sterile. Δcprα cells displayed wild-type mating efficiency when reconstituted with the wild-type CPRα gene. Hyphal production on filament agar was not affected in the Δcprα strain, indicating no significant role for CPRα in sensing environmental cues during haploid fruiting. The wild-type MATα CPRα strain produced abundant hyphae in response to synthetic MATa pheromone; however, the hyphal response to pheromone by Δcprα cells was significantly reduced. Exposure of wild-type cells to synthetic MATa pheromone for 2 h induced MFα pheromone expression, whereas unexposed cells showed only basal levels of the MFα transcript. The Δcprα cells, however, exhibited only basal levels of MFα message with or without pheromone exposure, suggesting that CPRα and MFα are components of the same signaling pathway

Transcriptional repression of CD44 suppressed the tumorigenecity and pSTAT3 activation.

<p>A: Tumorosphere formation assay of CD44 knock-down cells. A representative area was pictured from the tumorosphere cultures from MDA-MB-231 control RNA and CD44 transfected cells. B: Quantitative graph was presented for the tumor sphere formation assay of CD44 knock-down cells. C: Western analyses of CD44 shRNA transfected cells. Protein expression levels were examined for hTERT, pSTAT3, CD44 and CD24 from the CD44 knock-down cells. Downstream genes of pSTAT and hTERT levels were presented.</p

Schematic diagrams of STAT3-hTERT-CD44 autocrine signaling in breast cancer cells.

<p>STAT3 was found to bound CD44 and NF-kB concurrently. STAT3-NF-kB complex translocates into nucleus and binds to hTERT promoter and activated hTERT expression. Activated hTERT enhances CD44 expression in an autocrine manner in breast cancer stem cells.</p

Both MCF7-HER2 and MDA-MB-231 possess high CD44 (+) cellular populations.

<p>A: FACS profiles of MCF7_HER2. CD44-FITC was stained for MCF7-HER2 (passage 30) and subjected to FACS profiling. As shown in the figure, MCF7-HER2 possessed 76.72% of CD44 (+) cell population. B. CD44 FACS profiles of MDA-MB-231. CD44-FITC was stained for MDA-MB-231 and subjected FACS profiling. MDA-MB-231 possessed 99.05% of CD44 (+) cell population.</p