Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Image-guided sampling reveals increased stroma and lower glandular complexity in mammographically dense breast tissue

Mammographic density (MD) adjusted for age and body mass index (BMI) is a strong heritable breast cancer risk factor; however, its biological basis remains elusive. Previous studies assessed MD-associated histology using random sampling approaches, despite evidence that high and low MD areas exist within a breast and are negatively correlated with respect to one another. We have used an image-guided approach to sample high and low MD tissues from within individual breasts to examine the relationship between histology and degree of MD. Image-guided sampling was performed using two different methodologies on mastectomy tissues (n = 12): (1) sampling of high and low MD regions within a slice guided by bright (high MD) and dark (low MD) areas in a slice X-ray film; (2) sampling of high and low MD regions within a whole breast using a stereotactically guided vacuum-assisted core biopsy technique. Pairwise analysis accounting for potential confounders (i.e. age, BMI, menopausal status, etc.) provides appropriate power for analysis despite the small sample size. High MD tissues had higher stromal (P = 0.002) and lower fat (P = 0.002) compositions, but no evidence of difference in glandular areas (P = 0.084) compared to low MD tissues from the same breast. High MD regions had higher relative gland counts (P = 0.023), and a preponderance of Type I lobules in high MD compared to low MD regions was observed in 58% of subjects (n = 7), but did not achieve significance. These findings clarify the histologic nature of high MD tissue and support hypotheses regarding the biophysical impact of dense connective tissue on mammary malignancy. They also provide important terms of reference for ongoing analyses of the underlying genetics of MD.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Aberrant fibroblast growth factor receptor signaling in bladder and other cancers

Fibroblast growth factors (FGFs) are potent mitogens, morphogens, and inducers of angiogenesis, and FGF signaling governs the genesis of diverse tissues and organs from the earliest stages. With such fundamental embryonic and homeostatic roles, it follows that aberrant FGF signaling underlies a variety of diseases. Pathological modifications to FGF expression are known to cause salivary gland aplasia and autosomal dominant hypophosphatemic rickets, while mutations in FGF receptors (FGFRs) result in a range of skeletal dysplasias. Anomalous FGF signaling is also associated with cancer development and progression. Examples include the overexpression of FGF2 and FGF6 in prostate cancer, and FGF8 overexpression in breast and prostate cancers. Alterations in FGF signaling regulators also impact tumorigenesis, which is exemplified by the down-regulation of Sprouty 1, a negative regulator of FGF signaling, in prostate cancer. In addition, several FGFRs are mutated in human cancers (including FGFR2 in gastric cancer and FGFR3 in bladder cancer). We recently identified intriguing alterations in the FGF pathway in a novel model of bladder carcinoma that consists of a parental cell line (TSU-Pr1/T24) and two sublines with increasing metastatic potential (TSU-Pr1-B1 and TSU-Pr1-B2), which were derived successively through in vivo cycling. It was found that the increasingly metastatic sublines (TSU-Pr1-B1 and TSU-Pr1-B2) had undergone a mesenchymal to epithelial transition. FGFR2IIIc expression, which is normally expressed in mesenchymal cells, was increased in the epithelial-like TSU-Pr1-B1 and TSU-Pr1-B2 sublines and FGFR2 knock-down was associated with the reversion of cells from an epithelial to a mesenchymal phenotype. These observations suggest that modified FGF pathway signaling should be considered when studying other cancer types.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44HI/CD24lO/-stem cell phenotype in human breast cancer

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44highCD24low. Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.

Epithelial-to-mesenchymal transitions and circulating tumor cells

Epithelial-to-mesenchymal transition (EMT) phenomena endow epithelial cells with enhanced migratory and invasive potential, and as such, have been implicated in many physiological and pathological processes requiring cell migration/invasion. Although their involvement in the metastatic cascade is still a subject of debate, data are accumulating to demonstrate the existence of EMT phenotypes in primary human tumors, describe enhanced metastatic potential of EMT derivatives in animal models, and report EMT attributes in circulating tumor cells (CTCs). The relationships between EMT and CTCs remain largely unexplored, and we review here in vitro and in vivo data supporting a putative role of EMT processes in CTC generation and survival.

Carcinoma ex pleomorphic adenoma : a comprehensive review of clinical, pathological and molecular data

Carcinoma ex pleomorphic adenoma (Ca ex PA) is a carcinoma arising from a primary or recurrent benign pleomorphic adenoma. It often poses a diagnostic challenge to clinicians and pathologists. This study intends to review the literature and highlight the current clinical and molecular perspectives about this entity. The most common clinical presentation of CA ex PA is of a firm mass in the parotid gland. The proportion of adenoma and carcinoma components determines the macroscopic features of this neoplasm. The entity is difficult to diagnose pre-operatively. Pathologic assessment is the gold standard for making the diagnosis. Treatment for Ca ex PA often involves an ablative surgical procedure which may be followed by radiotherapy. Overall, patients with Ca ex PA have a poor prognosis. Accurate diagnosis and aggressive surgical management of patients presenting with Ca ex PA can increase their survival rates. Molecular studies have revealed that the development of Ca ex PA follows a multi-step model of carcinogenesis, with the progressive loss of heterozygosity at chromosomal arms 8q, then 12q and finally 17p. There are specific candidate genes in these regions that are associated with particular stages in the progression of Ca ex PA. In addition, many genes which regulate tumour suppression, cell cycle control, growth factors and cell-cell adhesion play a role in the development and progression of Ca ex PA. It is hopeful that these molecular data can give clues for the diagnosis and management of the disease.

Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-θ, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes α, μ, and π was studied with affnity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes α, μ, and π but also of enhancing UDGPT and GST-θ. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.

Fine-needle aspiration cytology of Merkel cell carcinoma-A review of 69 cases

Background This study reviewed the clinical presentation, cytologic findings and the immunophenotype of 69 Merkel Cell Carcinoma (MCC) cases sampled by FNA. Methods Demographic and clinical data, the cytology findings and results of ancillary testing were reviewed. Results Median patient age was 78 years (37 – 104) with a 1:1.8 female to male ratio. The most common FNA sites sampled included lymph nodes in the neck, the axillary region, the inguinal region and the parotid gland. Most patients had a history of MCC (68%) &/or non-MCC malignancy (70%). The common cytologic pattern was a cellular smear with malignant cells arranged in a dispersed pattern with variable numbers of disorganised groups of cells. Cytoplasm was scant or absent and nuclei showed mild to moderate anisokaryosis, stippled chromatin, inconspicuous nucleoli and nuclear molding. Numerous apoptotic bodies were often present. Cell block samples (28 cases) were usually positive for cytokeratins in a perinuclear dot pattern, including 88% of cases with CK20 positivity. CD56 was the most sensitive (95%) neuroendocrine marker on cell blocks and was also positive with flow cytometry in 9 cases tested. Conclusions MCC is most commonly seen in FNA specimens from the head and neck of elderly patients, often with a history of previous skin lesions. Occasional cases present in younger patients and some may be mistaken for other round blue cell tumors, such as lymphoma. CD 56 may be a useful marker in cell block preparations and in flow cytometric analysis of MCC.

Diagnostic potential of saliva : current state and future applications

BACKGROUND: Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. CONTENT: As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. SUMMARY: Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance. (C) 2011 American Association for Clinical Chemistry

Diagnostic potential of saliva : state of the art and future applications [Potenziale diagnostico della saliva: stato dell’arte e applicazioni future]

Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.

High-yeld RNA-extraction method for saliva

BACKGROUND: The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS: Oral whole saliva (200 mu L) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding beta-actin ("housekeeping" gene) and histatin (a salivary gland-specific gene). RESULTS: The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89 -7.1 mu g) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at -80 degrees C for >2 years. CONCLUSIONS: Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.