Expression analysis of mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2) in more than 300 human tumors and matching normal tissues reveals their co-expression in gynecologic malignancies

BACKGROUND: Mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2), two members of the secretoglobin superfamily, are known to be co-expressed in breast cancer, where their proteins form a covalent complex. Based on the relatively high tissue-specific expression pattern, it has been proposed that the mammaglobin A protein and/or its complex with lipophilin B could be used in breast cancer diagnosis and treatment. In view of these clinical implications, the aim of the present study was to analyze the expression of both genes in a large panel of human solid tumors (n = 309), corresponding normal tissues (n = 309) and cell lines (n = 11), in order to evaluate their tissue specific expression and co-expression pattern. METHODS: For gene and protein expression analyses, northern blot, dot blot hybridization of matched tumor/normal arrays (cancer profiling arrays), quantitative RT-PCR, non-radioisotopic RNA in situ hybridization and immunohistochemistry were used. RESULTS: Cancer profiling array data demonstrated that mammaglobin A and lipophilin B expression is not restricted to normal and malignant breast tissue. Both genes were abundantly expressed in tumors of the female genital tract, i.e. endometrial, ovarian and cervical cancer. In these four tissues the expression pattern of mammaglobin A and lipophilin B was highly concordant, with both genes being down-, up- or not regulated in the same tissue samples. In breast tissue, mammaglobin A expression was down-regulated in 49% and up-regulated in 12% of breast tumor specimens compared with matching normal tissues, while lipophilin B was down-regulated in 59% and up-regulated in 3% of cases. In endometrial tissue, expression of mammaglobin A and lipophilin B was clearly up-regulated in tumors (47% and 49% respectively). Both genes exhibited down-regulation in 22% of endometrial tumors. The only exceptions to this concordance of mammaglobin A/lipophilin B expression were normal and malignant tissues of prostate and kidney, where only lipophilin B was abundantly expressed and mammaglobin A was entirely absent. RNA in situ hybridization and immunohistochemistry confirmed expression of mammaglobin A on a cellular level in endometrial and cervical cancer and their corresponding normal tissues. CONCLUSION: Altogether, these data suggest that expression of mammaglobin A and lipophilin B might be controlled in different tissues by the same regulatory transcriptional mechanisms. Diagnostic assays based on mammaglobin A expression and/or the mammaglobin A/lipophilin B complex appear to be less specific for breast cancer, but with a broader spectrum of potential applications, which includes gynecologic malignancies

Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-α, IL-1β) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells

Effect of Cytoskeletal Disruption on Mechanotransduction of Hydrostatic Pressure by C3H10T1/2 Murine Fibroblasts

Cyclic hydrostatic pressure of physiological magnitude (< 10 MPa) stimulates chondrogenic differentiation of mesenchymal stem cells, but mechanotransduction mechanisms are not well understood. It was hypothesized that an intact cytoskeleton would be required for uninhibited mechanotransduction of hydrostatic pressure. Therefore we examined the effects of drugs which selectively interfere with actin and tubulin polymerization on pressure-induced upregulation of aggrecan and col2a1 (type II collagen) mRNA expression. C3H10T1/2 cells were cultured as pellets in either 4µM cytochalasin D or 4µM nocodazole and subjected to 3 days of cyclic hydrostatic compression (1 Hz, 5 MPa, 2 h per day). Phalloidin staining and indirect immunostaining with anti α-tubulin antibody confirmed disruption of microfilament and microtubule assemblies, respectively. Real time RT-PCR revealed that both drugs substantially lowered the basal level of aggrecan and col2a1 mRNA, but that neither drug prevented a pressure-stimulated increase in gene expression relative to the altered basal state. Thus upregulation of macromolecular gene expression by cyclic hydrostatic pressure did not require a completely intact cytoskeleton

Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results

Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.</p> <p>Methods</p> <p>Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.</p> <p>Results</p> <p>At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (<it>mam</it>) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).</p> <p>Conclusion</p> <p>This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the <it>mam </it>gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.</p

A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

Background: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. Principal Findings: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. Conclusions: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings sugges

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis