Aromatase expression in atypical ductal hyperplasia in women.

PURPOSE: To determine the levels of aromatase in atypical ductal hyperplasia (ADH) lesions, tissue surrounding the ADH, and in dense and non-dense normal breast tissue. We postulated that excess aromatase in breast tissue might, through production of increased estrogen, drive the carcinogenic process. Estrogens and their metabolites are thought to contribute to the development of breast cancer through estrogen receptor-mediated mechanisms and genotoxic effects of estrogen metabolites. ADH is a benign lesion of the breast which is associated with substantially increased risk for subsequent development of breast cancer. After 25 years, approximately 30% of women with ADH develop breast cancer. In women with three or more separate ADH lesions at the same time, 47% will develop breast cancer over that time period. Another important risk factor for breast cancer is the presence of mammographically dense breast tissue. METHODS: We utilized quantitative immunochemical analysis of aromatase in biopsy tissue to test this possibility. Previously published results comparing dense with non-dense breast tissue in normal women (Vachon et al. Breast Cancer Res Treat 125:243-252, 2011) were used for comparisons with ADH. A well-characterized histochemical H-score was employed for quantitative assessment of aromatase in the various tissue studied. RESULTS: The H-score of aromatase staining was statistically significantly higher (p = 0.003) in the ADH epithelium than surrounding epithelial tissue. In order of H-score from highest to lowest were ADH, issue surrounding ADH, dense normal and non-dense normal breast tissues. The levels of aromatase in a subset of women with ADH who went on to develop breast cancer were not higher than in women who did not. CONCLUSIONS: We suggest from these studies that overexpression of aromatase in breast tissue and its resultant increase in estradiol levels may contribute to the later development of breast cancer in women with ADH.

AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells.

Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-κB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.

Loss of type III transforming growth factor-beta receptor expression is due to methylation silencing of the transcription factor GATA3 in renal cell carcinoma.

Loss of transforming growth factor-beta receptor III (TbetaRIII) correlates with loss of transforming growth factor-beta (TGF-beta) responsiveness and suggests a role for dysregulated TGF-beta signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TbetaRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TbetaRIII expression in human cells. We localize GATA3's binding to a 10-bp region of the TbetaRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TbetaRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TbetaRIII expression during ccRCC progression.

Matrix metalloproteinase-10 is a critical effector of protein kinase Ciota-Par6alpha-mediated lung cancer.

Protein kinase Ciota (PKCiota) drives transformed growth of non-small cell lung cancer (NSCLC) cells through the Rho family GTPase Rac1. We show here that PKCiota activates Rac1 in NSCLC cells by formation of a PKCiota-Par6alpha complex that drives anchorage-independent growth and invasion through activation of matrix metalloproteinase-10 (MMP-10) expression. RNAi-mediated knockdown of PKCiota, Par6alpha or Rac1 expression inhibits NSCLC transformation and MMP-10 expression in vitro. Expression of wild-type Par6alpha in Par6alpha-deficient cells restores transformation and MMP-10 expression, whereas expression of Par6alpha mutants that either cannot bind PKCiota (Par6alpha-K19A) or couple to Rac1 (Par6alpha-DeltaCRIB) do not. Knockdown of MMP-10 expression blocks anchorage-independent growth and invasion of NSCLC cells and addition of catalytically active MMP-10 to PKCiota- or Par6alpha-deficient cells restores anchorage-independent growth and invasion. Dominant-negative PKCiota inhibits tumorigenicity and MMP-10 expression in subcutaneous NSCLC tumors. MMP-10 and PKCiota are coordinately overexpressed in primary NSCLC tumors, and tumor MMP-10 expression predicts poor survival in NSCLC patients. Our data define a PKCiota-Par6alpha-Rac1 signaling axis that drives anchorage-independent growth and invasion of NSCLC cells through induction of MMP-10 expression.

Microenvironmental regulators of tissue structure and function also regulate tumor induction and progression: the role of extracellular matrix and its degrading enzymes.

It is now widely accepted that elements of the cellular and tissue microenvironment are crucial regulators of cell behavior in culture and homeostasis in vivo, and that many of the same factors influence the course of tumor progression. Less well established is the extent to which extracellular factors actually cause cancer, and the circumstances under which this may occur. Using physiologically relevant three-dimensional culture assays and transgenic animals, we have explored how the environmental and architectural context of cells, tissues, and organs controls mammary-specific gene expression, growth regulation, apoptosis, and drug resistance and have found that loss of tissue structure is a prerequisite for cancer progression. Here we summarize this evidence and highlight two of our recent studies. Using mouse mammary epithelial cells, we show that exposure to matrix metalloproteinase-3 (MMP-3) stimulates production of reactive oxygen species (ROS) that destabilize the genome and induce epithelial-mesenchymal transition, causing malignant transformation. Using a human breast cancer progression series, we find that ADAM-dependent growth factor shedding plays a crucial role in acquisition of the malignant phenotype. These findings illustrate how normal tissue structure controls the response to extracellular signals so as to preserve tissue specificity and growth status.

Integrating naive Bayes models and external knowledge to examine copper and iron homeostasis in S. cerevisiae.

A novel suite of analytical techniques and visualization tools are applied to 78 published transcription profiling experiments monitoring 5,687 Saccharomyces cerevisiae genes in studies examining cell cycle, responses to stress, and diauxic shift. A naive Bayes model discovered and characterized 45 classes of gene profile vectors. An enrichment measure quantified the association between these classes and specific external knowledge defined by four sets of categories to which genes can be assigned: 106 protein functions, 5 stages of the cell cycle, 265 transcription factors, and 16 chromosomal locations. Many of the 38 genes in class 42 are known to play roles in copper and iron homeostasis. The 17 uncharacterized open reading frames in this class may be involved in similar homeostatic processes; human homologs of two of them could be associated with as yet undefined disease states arising from aberrant metal ion regulation. The Met4, Met31, and Met32 transcription factors may play a role in coregulating genes involved in copper and iron metabolism. Extensions of the simple graphical model used for clustering to learning more complex models of genetic networks are discussed.

The yeast frataxin homologue mediates mitochondrial iron efflux. Evidence for a mitochondrial iron cycle.

Mutations in the nuclear gene encoding the mitochondrial protein frataxin are responsible for the neurological disorder Friedreich ataxia (FA). Yeast strains with a deletion in the frataxin homologue YFH1 accumulate excess iron in mitochondria and demonstrate mitochondrial damage. We show that in the absence of YFH1, mitochondrial damage is proportional to the concentration and duration of exposure to extracellular iron, establishing mitochondrial iron accumulation as causal to mitochondrial damage. Reintroduction of YFH1 results in the rapid export of accumulated mitochondrial iron into the cytosol as free, non-heme bound iron, demonstrating that mitochondrial iron in the yeast FA model can be made bioavailable. These results demonstrate a mitochondrial iron cycle in which Yfh1p regulates mitochondrial iron efflux.

Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts.

Examination of the mechanism of intracellular iron recovery from lysosomally-degraded ferritin in vivo has been complicated by the continuous flux of cellular iron through ferritin molecules. Here we incubated human fibroblasts with cationic ferritin, a derivative of horse spleen ferritin, as a technique for delivering immunologically distinct ferritin molecules directly to lysosomes. Using this method, we found increased endogenous ferritin levels after the cellular degradation of cationic ferritin, demonstrating that cells can utilize lysosomal ferritin to produce increased cytosolic ferritin levels. Further, using an in vitro assay, we showed that isolated lysosomes degrade endogenous ferritin in a time- and temperature-dependent manner. These results are consistent with a model in which cytosolic ferritin is taken into the lysosomes and degraded. The solubilized iron from the ferric core could then be transported across the lysosomal membrane back into the cytosol.

Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast.

Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans. The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.

Makaluvamines, marine natural products, are active anti-cancer agents and DNA topo II inhibitors.

The makaluvamines were isolated from a sponge of the genus Zyzzya by following bioactivity against the human colon carcinoma cell line, HCT 116. These compounds have considerable cytotoxic activity. The makaluvamines appear to be acting through inhibition of DNA topoisomerase II. The compounds show enhanced toxicity toward a topoisomerase II-cleavable complex-sensitive cell line, they inhibit topoisomerase II decatenation of kinetoplast DNA in vitro. Makaluvamine C was shown to produce protein-linked DNA double-strand breaks, and makaluvamine A produced DNA double-strand breaks by neutral filter elution in a dose-dependent fashion similar to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). The makaluvamines also increased the life span of nude mice bearing solid tumors of human ovarian cancer cells.