Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focusses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice

Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.

Assessing the role of spatial correlations during collective cell spreading

Spreading cell fronts are essential features of development, repair and disease processes. Many mathematical models used to describe the motion of cell fronts, such as Fisher’s equation, invoke a mean–field assumption which implies that there is no spatial structure, such as cell clustering, present. Here, we examine the presence of spatial structure using a combination of in vitro circular barrier assays, discrete random walk simulations and pair correlation functions. In particular, we analyse discrete simulation data using pair correlation functions to show that spatial structure can form in a spreading population of cells either through sufficiently strong cell–to–cell adhesion or sufficiently rapid cell proliferation. We analyse images from a circular barrier assay describing the spreading of a population of MM127 melanoma cells using the same pair correlation functions. Our results indicate that the spreading melanoma cell populations remain very close to spatially uniform, suggesting that the strength of cell–to–cell adhesion and the rate of cell proliferation are both sufficiently small so as not to induce any spatial patterning in the spreading populations.

B-Raf mutation : a key player in molecular biology of cancer

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common.

Clinicopathological relevance of BRAF mutations in human cancer

BRAF represents one of the most frequently mutated protein kinase genes in human tumours. The mutation is commonly tested in pathology practice. BRAF mutation is seen in melanoma, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these cancers, various genetic aberrations of the BRAF proto-oncogene, such as different point mutations and chromosomal rearrangements, have been reported. The most common mutation, BRAF V600E, can be detected by DNA sequencing and immunohistochemistry on formalin fixed, paraffin embedded tumour tissue. Detection of BRAF V600E mutation has the potential for clinical use as a diagnostic and prognostic marker. In addition, a great deal of research effort has been spent in strategies inhibiting its activity. Indeed, recent clinical trials involving BRAF selective inhibitors exhibited promising response rates in metastatic melanoma patients. Clinical trials are underway for other cancers. However, cutaneous side effects of treatment have been reported and therapeutic response to cancer is short-lived due to the emergence of several resistance mechanisms. In this review, we give an update on the clinical pathological relevance of BRAF mutation in cancer. It is hoped that the review will enhance the direction of future research and assist in more effective use of the knowledge of BRAF mutation in clinical practice.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Advance Care Planning in palliative care : a systematic literature review of the contextual factors influencing its uptake 2008-2012

Background: Advance Care Planning is an iterative process of discussion, decision-making and documentation about end-of-life care. Advance Care Planning is highly relevant in palliative care due to intersecting clinical needs. To enhance the implementation of Advance Care Planning, the contextual factors influencing its uptake need to be better understood. Aim: To identify the contextual factors influencing the uptake of Advance Care Planning in palliative care as published between January 2008 and December 2012. Methods: Databases were systematically searched for studies about Advance Care Planning in palliative care published between January 2008 and December 2012. This yielded 27 eligible studies, which were appraised using National Institute of Health and Care Excellence Quality Appraisal Checklists. Iterative thematic synthesis was used to group results. Results: Factors associated with greater uptake included older age, a college degree, a diagnosis of cancer, greater functional impairment, being white, greater understanding of poor prognosis and receiving or working in specialist palliative care. Barriers included having non-malignant diagnoses, having dependent children, being African American, and uncertainty about Advance Care Planning and its legal status. Individuals’ previous illness experiences, preferences and attitudes also influenced their participation. Conclusion: Factors influencing the uptake of Advance Care Planning in palliative care are complex and multifaceted reflecting the diverse and often competing needs of patients, health professionals, legislature and health systems. Large population-based studies of palliative care patients are required to develop the sound theoretical and empirical foundation needed to improve uptake of Advance Care Planning in this setting.

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Nephrotoxicity and nephrocarcinogenicity of dinitrotoluene : new aspects to be considered

Technical dinitrotoluene (DNT) is a mixture of 2,4- and 2,6-DNT. In humans, industrial or environmental exposure can occur orally, by inhalation, or by skin contact. The classification of DNT as an 'animal carcinogen' is based on the formation of malignant tumors in kidneys, liver, and mammary glands of rats and mice. Clear signs of toxic nephropathy were found in rats dosed with DNT, and the concept was derived of an interrelation between renal toxicity and carcinogenicity. Recent data point to the carcinogenicity of DNT on the urinary tract of exposed humans. Between 1984 and 1997, 6 cases of urothelial cancer and 14 cases of renal cell cancer were diagnosed in a group of 500 underground mining workers in the copper mining industry of the former GDR and having high exposures to explosives containing technical DNT. The incidences of both urothelial and renal cell tumors in this group were 4.5 and 14.3 times higher, respectively, than anticipated on the basis of the cancer registers of the GDR. The genotyping of all identified tumor patients for the polymorphic enzymes NAT2, GSTM1, and GSTT1 identified the urothelial tumor cases as exclusively 'slow acetylates'. A group of 161 miners highly exposed to DNT was investigated for signs of subclinical renal damage. The exposures were categorized semi-quantitatively into 'low', 'medium', 'high', and 'very high'. A straight dose-dependence of the excretion of urinary biomarker proteins with the ranking of exposure was seen. Biomarker excretion (alpha1-microglobulin, glutathione S-transferases alpha and pi) indicated that DNT-induced damage was directed toward the tubular system. New data on DNT-exposed humans appear consistent with the concept of cancer initiation by DNT isomers and the subsequent promotion of renal carcinogenesis by selective damage to the proximal tubule. The differential pathways of metabolic activation of DNT appear to apply to the proximal tubule of the kidney and to the urothelium of the renal pelvis and lower urinary tract as target tissues of carcinogenicity.

Carcinogenicity and genotoxicity of ethylene oxide : new aspects and recent advances

Long-term inhalation studies in rodents have presented unequivocal evidence of experimental carcinogenicity of ethylene oxide, based on the formation of malignant tumors at multiple sites. However, despite a considerable body of epidemiological data only limited evidence has been obtained of its carcinogenicity in humans. Ethylene oxide is not only an important exogenous toxicant, but it is also formed from ethylene as a biological precursor. Ethylene is a normal body constituent; its endogenous formation is evidenced by exhalation in rats and in humans. Consequently, ethylene oxide must also be regarded as a physiological compound. The most abundant DNA adduct of ethylene oxide is 7-(2-hydroxyethyl)guanine (HOEtG). Open questions are the nature and role of tissue-specific factors in ethylene oxide carcinogenesis and the physiological and quantitative role of DNA repair mechanisms. The detection of remarkable individual differences in the susceptibility of humans has promoted research into genetic factors that influence the metabolism of ethylene oxide. With this background it appears that current PBPK models for trans-species extrapolation of ethylene oxide toxicity need to be refined further. For a cancer risk assessment at low levels of DNA damage, exposure-related adducts must be discussed in relation to background DNA damage as well as to inter- and intraindividual variability. In rats, subacute ethylene oxide exposures on the order of 1 ppm (1.83 mg/m3) cause DNA adduct levels (HOEtG) of the same magnitude as produced by endogenous ethylene oxide. Based on very recent studies the endogenous background levels of HOEtG in DNA of humans are comparable to those that are produced in rodents by repetitive exogenous ethylene oxide exposures of about 10 ppm (18.3 mg/m3). Experimentally, ethylene oxide has revealed only weak mutagenic effects in vivo, which are confined to higher doses. It has been concluded that long-term human occupational exposure to low airborne concentrations to ethylene oxide, at or below current occupational exposure limits of 1 ppm (1.83 mg/m3), would not produce unacceptable increased genotoxic risks. However, critical questions remain that need further discussions relating to the coherence of animal and human data of experimental data in vitro vs. in vivo and to species-specific dynamics of DNA lesions.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

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.