Human proximal tubule epithelial cells modulate autologous dendritic cell function

Background We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. Methods Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c+ BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. Results The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c+ BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. Conclusions Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

Fibroblast growth factor receptor inhibition synergizes with paclitaxel and doxorubicin in endometrial cancer cells

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.

Risk factors to predict the incidence of surgical adverse events following open or laparoscopic surgery for apparent early stage endometrial cancer: results from a randomised controlled trial

Aims: To identify risk factors for major Adverse Events (AEs) and to develop a nomogram to predict the probability of such AEs in individual patients who have surgery for apparent early stage endometrial cancer. Methods: We used data from 753 patients who were randomized to either total laparoscopic hysterectomy or total abdominal hysterectomy in the LACE trial. Serious adverse events that prolonged hospital stay or postoperative adverse events (using common terminology criteria 3+, CTCAE V3) were considered major AEs. We analyzed pre-surgical characteristics that were associated with the risk of developing major AEs by multivariate logistic regression. We identified a parsimonious model by backward stepwise logistic regression. The six most significant or clinically important variables were included in the nomogram to predict the risk of major AEs within 6 weeks of surgery and the nomogram was internally validated. Results: Overall, 132 (17.5%) patients had at least one major AE. An open surgical approach (laparotomy), higher Charlson’s medical co-morbidities score, moderately differentiated tumours on curettings, higher baseline ECOG score, higher body mass index and low haemoglobin levels were associated with AE and were used in the nomogram. The bootstrap corrected concordance index of the nomogram was 0.63 and it showed good calibration. Conclusions: Six pre-surgical factors independently predicted the risk of major AEs. This research might form the basis to develop risk reduction strategies to minimize the risk of AEs among patients undergoing surgery for apparent early stage endometrial cancer.

The utility of serum CA-125 in predicting extra-uterine disease in apparent early stage endometrial cancer

Objectives: To evaluate the clinical value of pre-operative serum CA125 in predicting the presence of extra-uterine disease in patients with apparent early stage endometrial cancer. Methods: Between October 6, 2005 and June 17, 2010, 760 patients were enrolled in an international, multicentre, prospective randomized trial (LACE) comparing laparotomy with laparoscopy in the management of endometrial cancer apparently confined to the uterus. This study is based on data from 657 patients with endometrial adenocarcinoma who had a pre-operative serum CA125 value, and was undertaken to correlate pre-operative serum CA125 with final stage. Results: Using a pre-operative CA-125 cutpoint of 30U/ml was associated with the smallest misclassification error (14.5%) using a multiple cross-validation method. Median pre-operative serum CA-125 was 14U/ml, and using a cutpoint of 30U/ml, 14.9% of patients had elevated CA-125 levels. Of 98 patients with elevated CA-125 level, 36 (36.7%) had evidence of extra-uterine disease. Of the 116 patients (17.7%) with evidence of extra-uterine disease, 31.0% had elevated CA-125 level. In univariate and multivariate logistic regression analysis, only pre-operative CA-125 level was found to be associated with extra-uterine spread of disease. Utilising a cutpoint of 30U/ml achieved a sensitivity, specificity, positive predictive value and negative predictive value of 31.0%, 88.5%, 36.7% and 85.7% respectively. Overall, 326/657 (49.6%) of patients had full surgical staging involving lymph node dissection. When analysis was limited to patients that had undergone full surgical staging, the outcomes remained essentially unchanged. Conclusions: Elevated CA-125 above 30U/ml in patients with apparent early stage disease is associated with a sensitivity of 31.0% and specificity of 88.5% in detecting extra-uterine disease. Pre-operative identification of this risk factor may assist to triage patients to tertiary centres and comprehensive surgical staging.

Improved surgical safety after laparoscopic compared to open surgery for apparent early stage endometrial cancer : results from a randomised controlled trial

AIM: To compare Total Laparoscopic Hysterectomy (TLH) and Total Abdominal Hysterectomy (TAH) with regard to surgical safety. METHODS: Between October 2005 and June 2010, 760 patients with apparent early stage endometrial cancer were enroled in a multicentre, randomised clinical trial (LACE) comparing outcomes following TLH or TAH. The main study end points for this analysis were surgical adverse events (AE), hospital length of stay, conversion from laparoscopy to laparotomy, including 753 patients who completed at least 6 weeks of follow-up. Postoperative AEs were graded according to Common Toxicity Criteria (V3), and those immediately life-threatening, requiring inpatient hospitalisation or prolonged hospitalisation, or resulting in persistent or significant disability/incapacity were regarded as serious AEs. RESULTS: The incidence of intra-operative AEs was comparable in either group. The incidence of post-operative AE CTC grade 3+ (18.6% in TAH, 12.9% in TLH, p 0.03) and serious AE (14.3% in TAH, 8.2% in TLH, p 0.007) was significantly higher in the TAH group compared to the TLH group. Mean operating time was 132 and 107 min, and median length of hospital stay was 2 and 5 days in the TLH and TAH group, respectively (p<0.0001). The decline of haemoglobin from baseline to day 1 postoperatively was 2g/L less in the TLH group (p 0.006). CONCLUSIONS: Compared to TAH, TLH is associated with a significantly decreased risk of major surgical AEs. A laparoscopic surgical approach to early stage endometrial cancer is safe.

Efficacy of oral or intrauterine device-delivered progestin in patients with complex endometrial hyperplasia with atypia or early endometrial adenocarcinoma : a meta-analysis and systematic review of the literature

Objectives: To investigate the efficacy of progestin treatment to achieve pathological complete response (pCR) in patients with complex atypical endometrial hyperplasia (CAH) or early endometrial adenocarcinoma (EC). Methods: A systematic search identified 3245 potentially relevant citations. Studies containing less than ten eligible CAH or EC patients in either oral or intrauterine treatment arm were excluded. Only information from patients receiving six or more months of treatment and not receiving other treatments was included. Weighted proportions of patients achieving pCR were calculated using R software. Results: Twelve studies met the selection criteria. Eleven studies reported treatment of patients with oral (219 patients, 117 with CAH, 102 with grade 1 Stage I EC) and one reported treatment of patients with intrauterine progestin (11 patients with grade 1 Stage IEC). Overall, 74% (95% confidence interval [CI] 65-81%) of patients with CAH and 72% (95% CI 62-80%) of patients with grade 1 Stage I EC achieved a pCR to oral progestin. Disease progression while on oral treatment was reported for 6/219 (2.7%), and relapse after initial complete response for 32/159 (20.1%) patients. The weighted mean pCR rate of patients with grade 1 Stage I EC treated with intrauterine progestin from one prospective pilot study and an unpublished retrospective case series from the Queensland Centre of Gynaecologic Oncology (QCGC) was 68% (95% CI 45- 86%). Conclusions: There is a lack of high quality evidence for the efficacy of progestin in CAH or EC. The available evidence however suggests that treatment with oral or intrauterine progestin is similarly effective. The risk of progression during treatment is small but longer follow-up is required. Evidence from prospective controlled clinical trials is warranted to establish how the efficacy of progestin for the treatment of CAH and EC can be improved further.

Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n=182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Gene expression profile of primary prostate epithelial and stromal cells in response to sulforaphane or iberin exposure

BACKGROUND: Broccoli consumption has been associated with a reduced risk of prostate cancer. Isothiocyanates (ITCs) derived from glucosinolates that accumulate in broccoli are dietary compounds that may mediate these health effects. Sulforaphane (SF, 4-methylsulphinylbutyl ITC) derives from heading broccoli (calabrese) and iberin (IB, 3-methylsulphinypropyl ITC) from sprouting broccoli. While there are many studies regarding the biological activity of SF, mainly undertaken with cancerous cells, there are few studies associated with IB. METHODS: Primary epithelial and stromal cells were derived from benign prostatic hyperplasia tissue. Affymetrix U133 Plus 2.0 whole genome arrays were used to compare global gene expression between these cells, and to quantify changes in gene expression following exposure to physiologically appropriate concentrations of SF and IB. Ontology and pathway analyses were used to interpret results. Changes in expression of a subset of genes were confirmed by real-time RT-PCR. RESULTS: Global gene expression profiling identified epithelial and stromal-specific gene expression profiles. SF induced more changes in epithelial cells, whereas IB was more effective in stromal cells. Although IB and SF induced different changes in gene expression in both epithelial and stromal cells, these were associated with similar pathways, such as cell cycle and detoxification. Both ITCs increased expression of PLAGL1, a tumor suppressor gene, in stromal cells and suppressed expression of the putative tumor promoting genes IFITM1, CSPG2, and VIM in epithelial cells. CONCLUSION: These data suggest that IB and SF both alter genes associated with cancer prevention, and IB should be investigated further as a potential chemopreventative agent.

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial–mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

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.