STAT3, HIF-1alpha, EPO and EPOR - signaling proteins in human primary ductal breast cancers

STAT3 upregulates expression of HIF-1 induced EPO. Receptor EPOR was reported to activate STAT3. Our study was aimed at demonstration of tissue immunoreactivities of those proteins and determination of their relationships in reference to clinicopathological variables of breast cancers. We detected STAT3, HIF-1alpha, EPO and EPOR in specimens of 76 human, female, ductal breast cancers by immunohistochemistry. STAT3 was detected in 38 of 76 cancers (50%). HIF-1alpha was found in 55 cases (72%). EPO positive tumors comprised 89% of all the cancers (68 cases). EPOR was also visualized in 55 cases (72%). Anti-HIF-1alpha and anti-STAT3 stained nuclei and cytoplasm of breast cancer cells in diffuse and finely granular fashion. Strong membranous expressions of EPO and EPOR were distributed in cytoplasmic and membranous granularity or diffuse staining. STAT3 correlated with HIF-1 in general (r=0.4012, p<0.0001) and in different patients' subgroups. STAT3 was significantly associated with EPO and EPOR in all the cancers (r=0.2370, p=0.039 and r=0.3336, p=0.003, respectively). Besides a correlation between STAT3 and EPOR in node negative ones, STAT3 wasn't related to EPO and EPOR in remaining subgroups. HIF-1alpha correlated with EPO and EPOR in most of analyzed groups. Immunoreactivity to EPO generally was associated with EPOR (r=0.3520, p=0.002). Statistically analyzed distributions of the proteins reflected functional dependences among STAT3, HIF-1alpha, EPO and EPOR in cellular signal conduction.     

Designer peptide antagonist of the leptin receptor with peripheral antineoplastic activity

The obesity hormone leptin has been implicated in the development and progression of different cancer types, and preclinical studies suggest that targeting leptin signaling could be a new therapeutic option for the treatment of cancer, especially in obese patients. To inhibit pro-neoplastic leptin activity, we developed leptin receptor (ObR) peptide antagonists capable of blocking leptin effects in vitro and in vivo. Our lead compound (Allo-aca), however, crosses the blood–brain-barrier (BBB), inducing undesirable orexigenic effects and consequent weight gain. Thus, redesigning Allo-aca to uncouple its central and peripheral activities should produce a superior compound for cancer treatment. The aim of this study was to generate novel Allo-aca analogs and test their biodistribution in vivo and anti-neoplastic activity in vitro in breast and colorectal cancer cells. Examination of several Allo-aca analogs resulted in the identification of the peptidomimetic, d-Ser, that distributed only in the periphery of experimental animals. d-Ser inhibited leptin-dependent-proliferation of ObR-positive breast and colorectal cancer cells in vitro at 1 nM concentration without exhibiting any partial agonistic activity. d-Ser efficacy was demonstrated in monolayer and three-dimensional cultures, and its antiproliferative action was associated with the inhibition of several leptin-induced pathways, including JAK/STAT3, MAPK/ERK1/2 and PI3K/AKT, cyclin D1, and E-cadherin. In conclusion, d-Ser is the first leptin-based peptidomimetic featuring peripheral ObR antagonistic activity. The novel peptide may serve as a prototype to develop new therapeutics, particularly for the management of obesity-related cancers.     

The expression of HIF-1α in primary hepatocellular carcinoma and its correlation with radiotherapy response and clinical outcome

The purpose of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) protein expression in hepatocellular carcinoma (HCC), and responses of abdominal metastatic lymph nodes (LNs) from HCC patients treated with external beam radiotherapy (EBRT). HIF-1α immunohistochemical staining was performed on tissue microarrays (TMAs) of primary HCC specimens from 69 HCC patients with abdominal LN metastases. All patients received abdominal metastatic LN EBRT at the Department of Radiation Oncology at Zhongshan Hospital. A receiver-operating characteristic (ROC)-based approach and logistical regression analysis were used to determine the predictive value of HIF-1α expression in primary tumors with HCC metastatic LN EBRT response. Kaplan–Meier curves and log-rank tests were used to analyze patient survival. Cox proportional hazards regression model was used to analyze independent prognostic factors. HIF-1α expression was correlated with blood hemoglobin (Hb: r = −0.280, P = 0.020), response of abdominal metastatic LNs to EBRT (r = 0.286, P = 0.017), locoregional recurrence (r = 0.278, P = 0.021), and cancer-specific deaths (r = 0.298, P = 0.013). HIF-1α expression was predictive of EBRT response of metastatic LNs [area under the curve (AUC): 0.646; 95% confidence interval (CI): 0.499–0.793; P = 0.047], locoregional recurrence (AUC: 0.657; 95% CI: 0.509–0.805; P = 0.049) and cancer-specific deaths (AUC: 0.671; 95% CI: 0.531–0.812; P = 0.035). Patients with tumors exhibiting high HIF-1α expression had significantly poorer overall survival (OS) than those with low tumor expression of HIF-1α (P = 0.016). Multivariate analysis showed that Hb (P = 0.035), vascular invasion (P = 0.026), Child-Pugh score (P < 0.001), intrahepatic tumor control (P < 0.001), and HIF-1α (P = 0.020) were independent prognosis factors for OS of HCC patients after receiving abdominal metastatic LN EBRT. HIF-1α expression in primary HCCs was associated with EBRT response of abdominal metastatic LNs and poor prognosis.     

Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

Introduction  

Leptin is a peptide hormone with a role in bone metabolism and rheumatic diseases. The subchondral bone tissue plays a prominent role in the pathophysiology of osteoarthritis (OA), related to abnormal osteoblast (Ob) differentiation. Although leptin promotes the differentiation of Ob under normal conditions, a role for leptin in OA Ob has not been demonstrated. Here we determined if endogenous leptin produced by OA Ob could be responsible for the expression of the abnormal phenotypic biomarkers observed in OA Ob.     

Drug Development-targeted Screening of Leptin Agonist Glycopeptides

A glycosylated dodecapeptide fragment corresponding to the hypothalamus-active cytokine leptin exhibits agonistic properties to the leptin receptor (ObR) in vitro and penetrates into the brain in vivo. In order to characterize the drug development potential of the lead peptide and to optimize it for pharmacological applicability, a series of biochemical screening assays were custom-tailored to the leptin/ObR system. To identify peptides that bind the extracellular domain of ObR, we characterized the optimal conditions for an ELISA-type assay where the leptin fragments were immobilized to the plates. With this technology we could identify low-dose binder peptidomimetics which, according to a comparison of the conventional cell proliferation assay and a measure of metabolically active cells, revealed that agonists identified by these cellular assays may not necessarily induce the expected growth characteristics in ObR expressing cells. The original glycopeptide lead displayed a 2 h half life in 25% diluted mouse serum but poor stability in mouse brain extract. Fifteen percent of the glycopeptide crossed a dual endothelial/astrocyte cell layer (representing an in vitro model of blood-brain-barrier) in 30 min, and the coexistence of the two cell types appeared necessary to quantify the level of brain accessibility. Finally, in an in vivo mouse model, a Cy5.5 labeled glycopeptide was more evenly distributed all over the body, including the brain, than a similarly labeled full-sized leptin protein.     

Exploring Leptin Antagonism in Ophthalmic Cell Models

Background

Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.

Methods and Results

The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.

Conclusions

Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.     

Evaluation of the leptin receptor in human spermatozoa

Background  

Leptin, a 167 amino acid peptide hormone, profoundly effects reproduction exerting its biological effects via interaction with the leptin receptor (ObR) which is widely expressed on peripheral tissues. In this study, we have attempted to assess leptin receptor expression in the spermatozoa of fertile males and those diagnosed with male factor infertility; both at the mRNA or protein levels.     

High expression of leptin receptor leads to temozolomide resistance with exhibiting stem/progenitor cell features in gliobalastoma

Glioblastoma is a highly aggressive malignant disease with notable resistance to chemotherapy. In this study, we found that leptin receptor (ObR)-positive glioblastoma cells were resistant to temozolomide (TMZ), and TMZ-resistant cells exhibited high expression of ObR. ObR can serve as a marker to enrich glioblastoma cells with some stem/progenitor cell traits, which explained the reason for TMZ resistance of ObR+ cells. STAT3-mediated SOX2/OCT4 signaling axis maintained the stem/progenitor cell properties of ObR+ cells, which indirectly regulated glioblastoma TMZ resistance. These findings gain insight into the molecular link between obesity and glioblastoma, and better understanding of this drug-resistant population may lead to the development of more effective therapeutic interventions for glioblastoma.     

Association of serum amyloid A levels with adipocyte size and serum levels of adipokines: Differences between men and women

The aim of this study was to characterize the association between adipocyte enlargement and circulating levels of serum amyloid A (SAA). Furthermore, we wanted to search for possible associations with measures of glycemic control and levels of circulating adipokines and/or inflammatory markers in men and women with a large range in body mass index. The study cohort consisted of 167 subjects, 114 non-diabetic and 53 with Type 2 diabetes. Adipocyte diameter as well as circulating levels of SAA, C-reactive protein (CRP), adiponectin, leptin, interleukin-6, tumor necrosis factor alpha, glucose and insulin were measured. Women had higher serum levels of SAA than men (p = 0.044). SAA levels were weakly but positively correlated with BMI (p = 0.043) and % body fat (p = 0.027) in all subjects as well as subcutaneous adipocyte diameter (p = 0.034) in women. Furthermore, in all subjects we found correlations between SAA levels and levels of CRP (p < 0.001), interleukin-6 (p < 0.001), leptin (p = 0.003), insulin (p = 0.006), HbA1c (p = 0.02) and HOMA-IR (p = 0.002). A majority of the correlations were strongest in women. In conclusion, serum levels of SAA are strongly correlated with serum levels of inflammatory markers as well as measures of glycemic control. There seems to be large sex differences in these associations suggesting that sex-specific factors need to be considered when analyzing SAA levels in relation to metabolic disease.     

Exogenous leptin promotes the recovery of regressed ovary in fasted ducks

The present study was undertaken to examine the effect of administered recombinant mouse leptin on the recovery of regressed ovary in fasted ducks. Twenty-eight ducks were divided into five groups: fed ad libitum (control; n = 5), fasted control (FC; n = 5), fasted + low dose of leptin (F + L; n = 5), fasted + medium dose of leptin (F + M; n = 5) and fasted + high dose of leptin (F + H; n = 3). All four fasted groups were fasted for 2 days and then ad libitum and the ducks were treated with leptin at doses of 0 (control and FC), 50 (F + L), 250 (F + M) and 1000 (F + H) μg/kg body weight/day on day 3–5. Results showed that a moderate dose of leptin (250 μg/kg body weight/day) injected during the re-feeding period: (i) promoted the recovery of the regressed ovary as evidenced by an increase in ovary weight and recovery of yellow hierarchical follicles; (ii) elevated the plasma 17β-estradiol (E₂) level; (iii) increased the mRNA levels of ovary follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and estrogen receptor-β (ER-β). Furthermore, the results also showed that a high dose of leptin (1000 μg/kg body weight/day) may have a negative effect on the recovery of the regressed ovary. In conclusion, this study indicates that, in ducks, leptin may be involved in the recovery of the regressed ovary caused by 2 days of fasting. This effect may be related to increased plasma E₂ levels and stimulation of the mRNA levels of ovarian FSHR, LHR and especially ER-β.     

Ten-year survival analysis for renal carcinoma patients treated with an autologous tumour lysate vaccine in an adjuvant setting

About 30% of renal cell carcinomas (RCC) will develop recurrence after surgery. Despite evidence for a significantly improved survival by autologous tumour cell vaccination therapy, the procedure has not become standard. Between August 1993 and December 1996, 1,267 RCC patients undergoing radical nephrectomy in 84 German hospitals were subsequently treated by autologous tumour cell vaccination therapy. The study group comprised 692 patients with complete follow-up (stages pT2-3, pNx-2, M0 based on the TNM classification, 4th edition). Subsequent propensity-score matching according to 7 defined criteria with 861 control patients undergoing nephrectomy alone without adjuvant treatment at the Carl-Thiem-Hospital Cottbus, resulted in 495 matched pairs. Overall and stage-specific survival rates were analysed after a median follow-up of 131 months. The 5- and 10-year overall survival (OS) rates were 80.6 and 68.9% in the vaccine group and 79.2 and 62.1% in the control group (p = 0.066). Patients with pT3 stage RCC revealed 5- and 10-year OS rates of 71.3 and 53.6% in the study group and 65.4 and 36.2% in the control group (p = 0.022). In multivariable analysis, patients in the vaccine group showed a significantly improved survival both in the whole study group (HR = 1.28, p = 0.030) and in the subgroup presenting with pT3 stage tumours (HR = 1.67, p = 0.011). Adjuvant treatment with autologous vaccination therapy resulted in a significantly improved overall survival in pT3 stage RCC patients, suggesting benefit especially in this subgroup. However, controlled clinical trials integrating the recent TNM classification and further risk constellations are required to define additional patient groups that may derive benefit from this treatment.     

Association of plasma leptin,pro‐inflammatory adipokines and cancer‐related fatigue in early‐stage breast cancer patients: A prospective cohort study

Cancer‐related fatigue (CRF) is subjective and has wide inter‐individual variability. Given that leptin is commonly associated with fatigue syndrome, its use as a potential biomarker for CRF is being investigated. The primary objective of this study was to evaluate the association between leptin and CRF in early‐stage breast cancer patients receiving chemotherapy. In a prospective cohort study, patients completed assessments at baseline (T1), during chemotherapy (T2) and after chemotherapy (T3). Levels of plasma leptin and adipokines were measured using a Luminex bead‐immunoassay and CRF was measured using the Multi‐Dimensional Fatigue Symptom Inventory‐Short Form (MFSI‐SF). Data were analysed longitudinally using a generalised estimating equation incorporating clinically relevant parameters and pro‐inflammatory adipokines. The analysis included 136 patients (mean age ± SD = 51.5 ± 8.8 years; 69.1% receiving anthracycline‐based chemotherapy). More patients experienced CRF at T3 (23.8%) than at T2 (13.8%) compared to baseline. An increase was observed in the median plasma leptin level at T2, followed by a decrease at T3 (T1: 4.07 ng/mL, T2: 4.95 ng/mL and T3: 3.96 ng/mL). In the multivariate model, the change in leptin levels over time was significantly associated with the total MFSI‐SF score (β = ?0.15, P = 0.003) after adjusting for the tumour necrosis factor‐α (TNF‐α) level, anxiety, depression, insomnia, age, menopausal status and type of chemotherapy. This is the first study to report leptin as a biomarker that predicts the onset of CRF over time. Future studies are required to validate the findings.     

Expression of a melanoma-tumor-associated antigen as demonstrated by a monoclonal antibody (D6.1) in cytopathologic preparations of human tumor cells from effusions and needle aspirates

Immunocytopathologic studies were performed on 79 fine needle aspiration biopsies (FNABs) and effusions from 13 melanomas and 57 other human neoplasms with the monoclonal antibody (MAb) D6.1 raised against a partially purified melanoma-tumor-associated antigen (MTAA). The purposes of these studies were (1) to evaluate the ability of MAb 6.1 to react with melanoma cells in cytopathologic preparations and (2) to define the spectrum of reactivity of MAb D6.1 in cytopathologic preparations of non-melanomas. Cytocentrifuge preparations of the cytopathologic specimens were permitted to react with the primary antibody and were then stained by the avidin-biotin-immunoperoxidase method. Thirteen of 13 FNABs of malignant melanomas exhibited staining reactivity with MAb D6.1. Among the nonmelanoma tumors tested, staining reactivity was observed in 30 of 57 specimens. Among specific neoplasms, staining was present in 5 of 11 adenocarcinomas of the breast, 2 of 7 ovarian adenocarcinomas and 5 of 6 metastatic adenocarcinomas from the colon. Among 17 lung cancers examined, staining was noted in 4 of 7 adenocarcinomas, 3 of 4 large-cell undifferentiated carcinomas and 2 of 3 poorly differentiated squamous-cell carcinomas. Two small-cell undifferentiated carcinomas and one carcinoid failed to stain. Three of three adenocarcinomas of the pancreas showed staining. Among the remaining neoplasms examined, one specimen each of carcinoma of the prostate and the cervix and one carcinoma of undetermined primary exhibited staining. Two malignant lymphomas did not stain. Staining of mesothelial cells was observed in three of nine benign effusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.