Increased expression and serum levels of the stromal cell-secreted protein periostin in breast cancer bone metastases.

Periostin, a matricellular protein, is overexpressed in the stroma of several cancers. The aim of our study was to investigate more specifically whether periostin expression is associated with bone metastases from breast cancer and to determine its source in the affected bone. Nude mice were inoculated with human MDA-B02 breast cancer cells. Bone metastases-bearing mice were treated with zoledronic acid-an antiresorptive drug-or vehicle. Bone metastases were examined for tumor- and stroma-derived periostin expression by quantitative polymerase chain reaction with human- and mouse-specific primers and immunohistochemistry. Serum periostin and conventional bone turnover markers were also measured. MDA-B02 cells did not express periostin both in vitro and in vivo. However, mouse-derived periostin was markedly overexpressed (eightfold) in metastatic legs compared to noninoculated mice. Serum periostin levels were also markedly increased in metastatic mice and correlated with in situ expression levels. Immunostaining showed that periostin derived from the environing stromal cells of bone metastasis. Bone turnover blockade by zoledronic acid markedly decreased osteolytic lesions but only slightly modulated serum periostin levels. Bone metastases from breast cancer induce overexpression of periostin by surrounding stromal cells. Periostin could be a biochemical marker of the early stromal response associated to breast cancer bone metastasis formation.

Development of a new ELISA for serum periostin: evaluation of growth-related changes and bisphosphonate treatment in mice.

Periostin is a gamma-carboxyglutamic acid protein preferentially expressed in periosteum and bone mesenchymal stem cells. Lack of a precise assay for measuring circulating levels impairs the investigation of its biological significance. We developed a new ELISA and studied changes of periostin levels both locally at the bone site and systemically in circulating blood during growth and after bisphosphonate-induced inhibition of bone remodeling in the mouse. The ELISA we developed is based on an affinity-purified polyclonal antibody that was raised against the C-terminal sequence of mouse periostin. Reproducibility, repeatability, precision, and accuracy tests met standards of acceptance. Serum periostin and levels of the bone turnover markers osteocalcin, PINP, CTX-I, and TRAP5b were measured in (1) 4-, 6-, 8-, 10-, and 12-week-old wild-type female Balb/c mice and (2) adult ovariectomized female Balb/c mice treated with zoledronic acid or vehicle. Serum periostin decreased during growth and stabilized from 8 weeks and older, its levels correlating with bone turnover markers. Immunohistochemistry in bones from different growth stages showed that periostin localized specifically at the sites of endochondral and intramembranous ossification, especially at the periosteal envelopes. Zoledronic acid induced a marked decrease in bone remodeling markers but did not alter serum periostin levels or periostin immunostaining pattern. The novel ELISA is highly specific and allows accurate and precise measurements of serum periostin levels in mice.

Assessment of circulating Dickkopf-1 with a new two-site immunoassay in healthy subjects and women with breast cancer and bone metastases.

The aim of our study was to investigate the sex- and age-related changes of serum Dickkopf-1 (Dkk-1), a soluble inhibitor of the Wnt signaling pathway, in healthy individuals and in patients with breast cancer (BC) and bone metastases (BM) using a new ELISA. Association of serum Dkk-1 with markers of bone turnover was also investigated. Serum Dkk-1 measurements were performed using a commercial sandwich ELISA in 150 healthy men, 175 healthy pre- and postmenopausal women (20-65 years), 22 women with BC and BM (mean age 63 years), and 16 women with BC and metastases at sites other than bone (mean age 53 years). Intra- and interassay coefficients of variation were below 7% and 12%, respectively. The detection limit was determined to be 0.018 microg/L. In healthy women and men, Dkk-1 did not change with age. Serum Dkk-1 modestly correlated with serum bone alkaline phosphatase (r = 0.19, P = 0.013) and serum C-terminal cross-linking telopeptide of type I collagen (r = 0.19, P = 0.014) in women but not in men. Dkk-1 levels were higher in women with BC and BM (5.57 +/- 5.50 microg/L) than in healthy age-matched controls (3.47 +/- 1.47 microg/L, P < 0.0001) and women with metastases at sites other than bone (3.57 +/- 1.66 microg/L, P = 0.0003). In conclusion, serum Dkk-1 is stable with age in healthy women and men and increases in patients with BC and BM. Measurements of circulating Dkk-1 with this new ELISA may be useful for the clinical investigation of patients with malignant bone diseases.

Circulating dickkopf-1 and radiological progression in patients with early rheumatoid arthritis treated with etanercept.

OBJECTIVE: Dickkopf-1 (Dkk-1) regulates bone remodeling in animal models of inflammatory arthritis, but its role in patients with rheumatoid arthritis (RA) remains unclear. METHODS: Baseline circulating Dkk-1 was measured in 113 patients with RA (< 3 yrs) who received etanercept (10 or 25 mg twice/week, n = 63) or methotrexate alone (n = 40) for 1 year. Progression was assessed by changes in radiological Sharp score. RESULTS: Increased Dkk-1 was associated with a higher risk of progression of bone erosion, independently of age, sex, baseline radiological damage, C-reactive protein, and disease activity in patients treated with etanercept. CONCLUSION: Dkk-1 may be an important mediator of bone erosion in patients with RA.

CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways.

BACKGROUND: CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. METHODS: The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. RESULTS: Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. CONCLUSION: These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively.

Coexpression of CD40 and CD40L on B lymphoma and carcinoma cells: an autocrine anti-apoptotic role.

To evaluate a possible autocrine role of CD40L, the expression and functional activity of CD40L on NHL and breast carcinoma cell lines were investigated. Using flow cytometry, CD40 was consistently detectable at the surface of all 5 NHL cell lines tested. CD40L expression was detectable at the surface of DAUDI (54%, MFI 47) and BJAB (12%, MFI 32) cell lines, and marginally on the RAJI cell line (7%, MFI 30), while 4 of 5 NHL cell lines (DAUDI, RAJI, BJAB, BL70) had detectable CD40L mRNA. CD40 was expressed on T47D and BT20 breast carcinoma cell lines while CD40L was detectable on T47D (93%, MFI 137) only. Both BT20 and T47D had detectable CD40 mRNA, while CD40L mRNA was detectable only in the T47D cell line. CD40, but not CD40L, was detectable on 6 renal, 1 prostatic and 1 colon carcinoma cell lines. CD40L expressed on tumor cells was functional, as shown by its capacity to decrease drug-induced apoptosis on CD40 expressing NHL and breast carcinoma cell lines, while irradiated CD40L negative cell line (BT20) had no effect. Blocking CD40L antibody abrogated the protective effect of irradiated CD40L positive T47D cell line against drug-induced apoptosis on BL70 cell line, confirming that CD40L is functional in the DAUDI and T47D cell lines. Importantly, blocking CD40L antibody increased drug-induced apoptosis in CD40L positive cell lines but had no effect on the CD40L negative cell lines. CD40L is expressed on CD40 positive B NHL and breast carcinoma cell lines and induces an autocrine antiapoptotic signal when cells are exposed to cytotoxic agents.