Loss of breast epithelial marker hCLCA2 promotes epithelial to mesenchymal transition and indicates higher risk of metastasis

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Long terminal repeats act as androgen-responsive enhancers for the PSA-Kallikrein locus

The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE:: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation. Copyright © 2009 by Lippincott Williams & Wilkins.

Prognostic factors in patients with advanced non-small cell lung cancer : data from the phase III FLEX study

The FLEX study demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). In the FLEX intention to treat (ITT) population, we investigated the prognostic significance of particular baseline characteristics. Individual patient data from the treatment arms of the ITT population of the FLEX study were combined. Univariable and multivariable Cox regression models were used to investigate variables with potential prognostic value. The ITT population comprised 1125 patients. In the univariable analysis, longer median survival times were apparent for females compared with males (12.7 vs 9.3 months); patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 compared with 1 compared with 2 (13.5 vs 10.6 vs 5.9 months); never smokers compared with former smokers compared with current smokers (14.6 vs 11.1 vs 9.0); Asians compared with Caucasians (19.5 vs 9.6 months); patients with adenocarcinoma compared with squamous cell carcinoma (12.4 vs 9.3 months) and those with metastases to one site compared with two sites compared with three or more sites (12.4 months vs 9.8 months vs 6.4 months). Age (<65 vs ≥65 years), tumor stage (IIIB with pleural effusion vs IV) and percentage of tumor cells expressing EGFR (<40% vs ≥40%) were not identified as possible prognostic factors in relation to survival time. In multivariable analysis, a stepwise selection procedure identified age (<65 vs ≥65 years), gender, ECOG PS, smoking status, region, tumor histology, and number of organs involved as independent factors of prognostic value. In summary, in patients with advanced NSCLC enrolled in the FLEX study, and consistent with previous analyses, particular patient and disease characteristics at baseline were shown to be independent factors of prognostic value. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. © 2012 Elsevier Ireland Ltd.

Cytotoxicity testing of burn wound dressings, ointments and creams : a method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). METHODS: We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. RESULTS: Seventeen products were tested. The least cytotoxic products were Melolite, White soft Paraffin and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet, Mepitel((R)), PolyMem((R)), DuoDerm((R)) and Xeroform. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream a moisturizer which contains the preservative Chlorocresol. CONCLUSION: This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Fetuin-A : a major fetal serum protein that promotes "wound closure" and scarless healing [Letter to the Editor]

Burn-wound healing is a dynamic, interactive process involving a number of cellular and molecular events and is characterized by inflammation, granulation tissue formation, re-epithelialization, and tissue remodeling (Greenhalgh, 2002; Linares, 2002). Unlike incisional-wound healing, it also requires extensive re-epithelialization due to a predominant horizontal loss of tissue and often heals with abnormal scarring when burns involve deep dermis. The early mammalian fetus has the remarkable ability to regenerate normal epidermis and dermis and to heal dermal incisional wounds with no signs of scarring. Extensive research has indicated that scarless healing appears to be intrinsic to fetal skin (McCallion and Ferguson, 1996; Ferguson and O’Kane, 2004). Previously, we reported a fetal burn model, in which 80-day-old ovine fetuses (gestation¼ 145–153 days) healed deep dermal partial thickness burns without scars, whereas postnatal lambs healed equal depth burns with significant scarring (Cuttle et al., 2005; Fraser et al., 2005). This burn model provided early evidence that fetal skin has the capacity to repair and restore dermal horizontal loss, not just vertical injuries.

Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin : evaluation in a rat segmental defect model

Objective To determine whether locally applied tobramycin influences the ability of recombinant human bone morphogenetic protein 2 (rhBMP-2) to heal a segmental defect in the rat femur. Methods The influence of tobramycin on the osteogenic differentiation of mesenchymal stem cells was first evaluated in vitro. For the subsequent, in vivo experiments, a 5-mm segmental defect was created in the right femur of each of 25 Sprague-Dawley rats and stabilized with an external fixator and four Kirschner wires. Rats were divided in four groups: empty control, tobramycin (11 mg)/absorbable collagen sponge, rhBMP-2 (11 μg)/absorbable collagen sponge, and rhBMP-2/absorbable collagen sponge with tobramycin. Bone healing was monitored by radiography at 3 and 8 weeks. Animals were euthanized at 8 weeks and the properties of the defect were compared with the intact contralateral femur. Bone formation in the defect region was assessed by dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing. Results Tobramycin exerted a dose-dependent inhibition of alkaline phosphatase induction and calcium deposition by mesenchymal stem cells cultured under osteogenic conditions. The inhibition was reversed in the presence of 500 ng/mL of rhBMP-2. Segmental defects in the rat femora failed to heal in the absence of rhBMP-2. Tobramycin exerted no inhibitory effects on the ability of rhBMP-2 to heal these defects and increased the bone area of the defects treated with rhBMP-2. Data obtained from all other parameters of healing, including dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing, were unaffected by tobramycin. Conclusions Although our in vitro results suggested that tobramycin inhibits the osteogenic differentiation of mesenchymal stem cells, this could be overcome by rhBMP-2. Tobramycin did not impair the ability of rhBMP-2 to heal critical-sized femoral defects in rats. Indeed, bone area was increased by nearly 20% in the rhBMP-2 group treated with tobramycin. This study shows that locally applied tobramycin can be used in conjunction with rhBMP-2 to enhance bone formation at fracture sites.

Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis : role of fibroblast growth factor receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice : real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

LCC15-MB : a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 μM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Assays for the study of human cancer cell invasion and metastasis

Two in vitro and two in vivo assays for the study of human cancer invasion and metastasis are described. The assays include in vitro invasiveness through an artificial basement membrane (Matrigel®), invasiveness and metastasis in nude mice of subcutaneously injected LacZ-transduced human tumor cells, in vitro adherence to basement membrane components, and LacZ-transduced human cancer cells injected intravenously into nude mice. In studies of the processes involved in human cancer cell invasion and metastasis, these four assays were found to be complementary, and thus provide a set of test systems for preclinical screening of agents which interfere with these processes.

GATA3 : a promising marker for metastatic breast carcinoma in serous effusion specimens

Background The utility of GATA3 as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. Methods Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from breast (30), female genital tract (13), gastrointestinal tract (7), lung adenocarcinoma (9), pancreas (1), kidney (1) and bladder (1). The breast carcinoma cases included 15 ductal carcinomas, 8 lobular carcinomas and in 7 the histology sub-type was not available. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and GCDFP-15 to compare sensitivity with GATA3. Results Positive nuclear staining for GATA3 was present in 90% (27/30) of metastatic breast carcinoma specimens. All non-breast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases showed weak positivity of benign lymphoid cells. Staining results were unambiguous as all positive cases showed intense nuclear staining in >50% of tumor cells. Mammaglobin (57%; 17/30) and GCDFP-15 (33%; 10/30) were less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only one additional case to those stained with GATA3. Conclusions GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration.